THERAPEUTIC COMPOSITION WITH NANOSCALE ACTIVATION AGENTS Field Of The Invention The present invention is directed toward specially engineered encapsulation vesicles designed for controlled release of therapeutic agents, and more particularly encapsulation vesicles such as liposomes, that are associated with an activation agent that is selectively activatable by an activation condition, and which may be capable of self assembly.

Background Of The Invention Liposomes are one type of encapsulation vesicle and are comprised of small vesicular sacs that resemble tiny cells. These sacs have an aqueous or hydrophillic interior volume separated generally by a hydrophobic durable bilayer membrane. Both water- soluble drugs and insoluble drugs can, therefore, be incorporated into these vesicles.

Depending upon the production process used, these vesicles may comprise a single membrane (unilamellar) or several membranes (multilamellar). This makes construction of such vesicles quite flexible. In addition, the typical size of the liposomes can range from 0.05 to several micrometers in diameter. The variation in size makes these vesicles an effective delivery vehicle for a variety of cellular targets.

Since their discovery, more than 35 years ago, liposomes have been used in a variety of ways to deliver a variety of different drugs. The prospect of targeting liposomes to cancer or tumor sites generated a considerable excitement in medical research in the 1960's and 1970's. Early liposome formulations, however, were no more effective than actual administration of drug. For instance, clinical studies using doxorubicin- containing liposomes showed little improvement in antitumor activity. The lack of improved antitumor activity was largely due to the fact that liposomes were unstable in blood and released a good portion of their bioactive contents as a consequence of rapid binding of plasma proteins (opsonization). Exposure of liposomal preparations to normal plasma proteins showed evidence of hundreds of bands of these proteins called"opsins"bound to the outer surface of the lipsome. These proteins would then act to mark the liposome as a foreign body for removal from the blood stream.

Secondly, liposomes that survived the destabilization process were immediately

sequestered by the fixed macrophages in the spleen and liver (the mononuclear phagocyte system (MPS)). Once internalized by macrophages, the liposomes were destroyed. The combination of instability and rapid uptake by the MPS system severely limited the use of liposomes as drug delivery systems (DOXIL Clinical Series Vol. 1, No. 1,1997).

Researchers recognized a need to improve liposome circulation times and to lower detection by the body's immune defenses. Comparisons were made to erythrocytes and other similar cells that circulate for extended periods of time before degradation by macrophages. By the late 1980's, it was determined that red blood cells have a thick coat of carbohydrate on their surface and this allows them to circulate for <BR> <BR> extended periods of time (Allen, T. M., Chonn, A. "Large Unilamellar liposomes with low uptake by the reticuloendothelial system". FEBS Lett. 1987; 223: 42-46). As a result, researchers were able to graft MPEG (methoxypolyethylene glycol), a hydrophillic polymer, onto the surface of the liposomes. These new liposomes were found to have extended longevity in plasma (Papahadjopouos, D., Allen, T. M., <BR> <BR> Gabizon, A. , Barenholz, Y. ,"Optimization and Upscaling of Doxorubicin-containing liposomes for clinical use. Journal of Pharmaceutical Sciences Vol. 79, No. 12, December 1990). These liposomes are available commercially from ALZA Corporation, Mountain View, CA, and were shown to stably encapsulate doxorubicin, recirculate for periods of several days after injection without releasing drug, penetrate into tumor cells, and release encapsulated drug within the tumor. The long residence times of the MPEG or PEG-coated liposomes may be explained by the steric stabilization effect provided by the MPEG or PEG molecules on the surface of the vesicles. In other words, the liposome surface comprises a protective hydrophillic layer that prevents interaction of the plasma components with the liposomes. As a result, PEG or MPEG-coated liposomes may circulate longer in the blood stream.

Even today, the mechanism of why these liposomes work effectively remains unclear.

However, the actions within tumors may help explain. For instance, the liposome is quite small (the average is approximately 100 nm) and this allow for optimized drug carrying and circulation time. In addition, most solid tumors exhibit unique pathoanatomic features, such as extensive angiogenesis, hyperpermeable and defective architecture, impaired lymphatic drainage, and greatly increased production

of mediators that enhance vascular permeability. These conditions, therefore, allows for MPEG or PEG-coated liposomes to extravasate in solid tumors through defects present in the endothelial barriers of newly forming blood vessels. In addition, inflammatory tissue and tissues with local infections also contain vasculature with greatly enhanced permeability and therefore have been shown to be targets for efficient liposome extravasation. Extravasation of these and other type liposomes probably occurs between gaps and other similar spaces that allow the liposomes to lodge themselves between tumor cells. Once positioned in place, it is believed that the enclosed drug material is released either by leakage or by liposome degradation caused by enzymes such as phospholipases (Working, P. K. , Newman, M. S. , Huang,<BR> S. K. , et al."Pharmacokinetics, biodistribution and therapeutic efficacy of doxorubicin encapsulated in Stealth@ liposomes (DOXIL@). Liposomes Res. 1994 ; 4: 667-687).

Although there is no solid evidence, it is postulated that the release of drugs into tumor cells probably occurs over a period of days and possibly weeks. If this hypothesis holds true, then the tumors would be exposed to high concentrations of drugs for prolonged periods of time, thus enhancing the efficacy of the chemotherapeutic effects of doxorubicin.

However, these systems suffer from a few important limitations. First of all, the actual dosage levels and delivery of drug concentrations remains unclear. Without knowledge of the delivery mechanism or dosage effect, treatments remain fairly crude on a"hit or miss"approach. In addition, degradation is likely to be incomplete in some vesicles, or not at all. Therefore, the efficiency and delivery regulation could be overall improved. In addition, exposure of tumor tissue to drugs over time often results in resistance of the tumor to the drugs. This occurs through exposure and rapid mutation of tumor cells during stages of metastasizing. Therefore, there is a strong need to provide a more precise method for high dosing of drug in targeted areas at prescribed time intervals to avoid potential drug resistance problems. There is also the need to be able to more precisely and predictably control the delivery of drugs or to deliver the drugs more quickly to defined locations to minimize overall complications, development of drug resistant cells and to lower side effects in the patient.

In contrast, non-targeted activation agents are being used and developed to treat bacterial, viral and other diseases. Certain activation agents such as pore forming agents or organic nanotubes are being developed to address drug resistance problems in bacteria and other microorganisms. Limited toxicity studies in mice have shown that some of these self-assembling materials may be effective for in vivo application (Bong, D. T, et al.,"Self-Assembling Organic Nanotubes, Angew. Chem. Int. Ed.

2001,40, 988-1011). These activation agents, can be easily synthesized, are flexible in design and can self-assemble. More recently, activation agents have been designed to be photoswitchable. Some of these activation agents have been shown to be effective in thin films or as transmembrane channels or ionophores. It has also been suggested that these activation agents may be effective in controlled release applications (Vollmer, M. S. , et al, "Photoswitchable Hydrogen Bonding in Self- Organized Cylindrical Peptide Systems, Angew. Chem. Int. Ed. 1999, 38, No. 11; Bong, D. T, et al.,"Self-Assembling Organic Nanotubes, Ange. Cliem. lilt. Ed. 2001, 40,988-1011 ; Sanchez-Quesada et al. ,"Cyclic Peptides as Molecular Adapters for a Pore forming Protein", Am. Chem. Soc., Vol. 122, No. 48,2000 ; Fernandez-Lopez et al. , "Antibacterial Agents Based on the cyclic D, L-a-peptide architecture", Nature, Vol. 412, July 26,2000). These activation agents, however, suffer from a few limitations as therapeutics. First of all, the activation agents can not be delivered site specifically and remain questionable regarding overall efficacy, and ability to self- assemble in vivo. Secondly, the naked dosing of large amounts of activation agents to patients is likely to cause severe immunological or toxicological problems. Third, in these forms, these activation agents do not facilitate controlled delivery and release of a bioactive agent from an encapsulation vesicle.

Therefore, there is a strong need to provide a therapeutic composition or method for delivering bioactive agents site specifically in vivo with little toxicological or immunological response. The present invention satisfies these and other described needs.

Summary Of The Invention The present invention relates to a composition of matter for therapeutic treatment of humans and other mammals. Basically, the therapeutic composition of the present

invention comprises an encapsulation vesicle such as a liposome, an activation agent that is activated in response to an activation condition, and a bioactive agent that is selectively released by operation of the activation agent in the encapsulation vesicle.

The activation agent may be capable of self assembly and integration into the vesicle membrane, or may reside on the vesicle surface, or may be completely contained in the vesicle. The self assembling activation agent is capable of undergoing a change in structure or gating to selectively release a bioactive agent from the vesicle.

Fundamentally, the self assembling activation agent responds to the activating condition by selectively altering the permeability of the vesicle. Importantly, the alteration in permeability of the vesicle, application of the activation condition or the activation agent, does not achieve release of the agent by destruction or degradation of the vesicle. Instead, the agent responds to the activation condition to permit controlled and targeted release of the agent without destruction of the vesicle.

In one embodiment, The encapsulation vesicle retains structural integrity and controllable release functionality by virtue of the self assembled activation agent within its membrane. To increase the targeting capability, the encapsulation vesicle may optionally allow the attachment of a targeting ligand and/or enclose a bioactive agent that is site specific in function.

As noted above, the activation agent is capable of being activated by an activation condition, and the activation must result in an alteration of the properties of the vesicle that contain or release the bioactive agent. For instance, the activation condition may be light and the agent may be photoswitchable. In this example, the activation agent responds to light by changing conformation, structure, or activity in such a way that the vesicle is caused to release the bioactive agent. Selective or targeted application of the activation condition provides the ability to control and localize the release of the bioactive agent by applying the condition to a specific site within the body, or at a particular time, when release of the bioactive agent is desired.

Moreover, to enhance the targeting capability, a targeting ligand may also be associated with the encapsulation vesicle to concentrate the vesicle and the active agent at a target site, such as a tissue or organ. Typically, this occurs prior to the application of the activation condition. Furthermore, the activating agent is associated with the vesicle by way of a physical association that is non-reactive to the host. This

association provides the ability to avoid detection and removal by the hosts reticuloendothelial system. Due to different available structures for the activation agent, the"association"with the vesicle comprise simple containment within the vesicle membrane, such as certain of the pore forming agents described below, integration into the vesicle membrane, such as the organic nanotubes described below, or attachment to the vesicle membrane surface.

The invention also provides methods for therapeutic treatment using the composition of the invention. The composition is administered to the patient where it is taken up or extravasated by the necrotic tissues. The encapsulation vesicle may be designed so that the activation agent remains in the encapsulation vesicle and is not incorporated into other cells or cell membranes using fusion, phagocytosis, endocytosis or other similar mechanisms.

The method for therapeutic treatment may also comprises contacting a cell membrane with a therapeutic composition that comprises an encapsulation vesicle and an activation agent, such as an organic nanotube in the encapsulation vesicle, and allowing the cell membrane to incorporate the therapeutic composition so that the activation agent of the therapeutic composition may be activated. Importantly, the method includes application of an activation condition that causes the membrane vesicle to fuse with a target cell to deliver the bioactive agent. Thus, the method includes administering the engineered vesicles of the invention, applying the activation condition, and achieving fusion of the vesicle to a target tissue.

Brief Description Of The Drawings FIG. 1 shows a schematic representation of an encapsulation vesicle with a self assembled activation agent incorporated into the vesicle membrane.

FIG. 1 A is an enlarged portion of FIG. 1 showing the activation agent and how it may form an intramolecular pore.

FIG. 1B is an enlarged portion of FIG. 1 showing the activation agent and how it may form a transmembrane barrel stave.

FIG. 2 shows a schematic representation of an encapsulation vesicle with protection layer (s) over the activation agents.

FIG. 3 shows the therapeutic method for using the compositions of the present invention.

FIG. 4A shows a schematic representation of the first step of a fusion process using the therapeutic composition of the present invention.

FIG. 4B shows a schematic representation of the second step of a fusion process using the therapeutic composition of the present invention.

FIG. 4C shows a schematic representation of the third step of a fusion process using the therapeutic composition of the present invention.

FIG. 4D shows a schematic representation of a fourth step of a fusion process using the therapeutic composition of the present invention.

FIG. 5 shows a schematic representation of the fusion process using an encapsulation vesicle, an activation agent and a seringe portion of a diphtheria toxin.

FIG. 6 shows a plan view representation of a photoswitchable nanotube.

FIG. 7 shows a more detailed schematic representation of the photoswitchable nanotube shown in FIG. 6.

Description Of The Specific Embodiments Compositions and methods for therapeutic or diagnostic treatment are provided. In the described methods the therapeutic composition may be used to contact a cell membrane. The cell membranes may be in vitro or in vivo and include both pathogenic and nonpathogenic cells unless clearly stipulated otherwise.

Before describing the invention in further detail, it is to be understood that the invention is not limited to the particular embodiments of the invention described

below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

In this specification and the appended claims, the singular forms"a","an"and"the" include plural reference unless the context clearly dictates otherwise.

"Activate"or"activate by an activation condition"refers to the application of physical, chemical or biochemical conditions or processes that will cause an activation agent to open, close, open and close, open or close, degrade, release a bioactive agent through or by the activation agent, release one or more molecules that may be photodynamically activated or activated by other activating conditions. For instance, an activation agent may be activated by an external light source or laser to open and release a bioactive agent.

"aHL-K8A"refers to a mutant hemolysin protein produced by replacing the lysine (K) at position 8 in the amino acid sequence with arginine (A). aHL-H5M refers to a mutant hemolysin protein produced by replacing the histidine (H) at position 5 in the amino acid sequence with methionine (M). aHL (1-172@132-293) refers to a particular mutant a-hemolysin protein that has been produced using recombinant DNA techniques.

R104C refers to the replacement of arginine (R) 104 in the a-hemolysin protein with cysteine (C).

K168C, refers to the replacement of lysine (K) 168 in the a-hemolysin protein with cysteine (C).

D183C refers to the replacement of aspartate (D) 183in the a-hemolysin protein with cysteine (C).

E11C refers to the replacement of glutamate (E) 11 in the a-hemolysin protein with cysteine (C).

"Bioactive agent"refers to a substance that may be used in connection with an application that is therapeutic or diagnostic, such as, for example, in methods for diagnosing the presence or absence of a disease in a patient and/or methods for treatment of a disease in a patient. The term also refers to a substance that is capable of exerting a biological effect in vitro or in vivo. The bioactive agents may be neutral, positively or negatively charged. Exemplary bioactive agents include for example prodrugs, targeting ligands, diagnostic agents, pharmaceutical agents, drugs, synthetic organic molecules, proteins, peptides, vitamins, steroids, steroid analogs and genetic material.

"Biocompatible"refers to materials that are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic responses and diseased states.

"Biomolecule"refers to molecules derived from a biological organism or source. For example, biomolecules may include and not be limited to proteins, peptides, amino acids, nucleotides, nucleosides, polynucleotides, carbohydrates, lipids, sphingolipids, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), tRNA, mRNA, derivatives or these materials, collagen, fibrinogen, antibodies and other well known materials from biological organisms.

"Carrier"refers to a pharmaceutically acceptable vehicle, which is a nonpolar, hydrophobic solvent, and which may serve as a reconstituting medium. The carrier may be aqueous based or organic based. Carriers include, inter alia, lipids, proteins, polyscaccharides, sugars, polymers, copolymers, and acrylates.

"Cell"refers to any one of the minute protoplasmic masses that make up organized tissue, comprising a mass of protoplasm surrounded by a membrane, including nucleated and unnucleated cells and organelles.

"Cell membrane"refers the commonly described lipid based exterior boundary of a cell. The cell membrane may or may not comprise proteins or receptors.

"Diseased cell", "pathogenic cell"or"pathological cell"refers to any cell that fails to operate in its naturally occurring condition or normal biochemical fashion. These cells should be capable of causing disease. For instance, the word shall include cells that are subject to uncontrolled growth, cellular mutation, metastasis or infection. The term shall also include cells that have been infected by a foreign virus or viral particle, bacteria, bacterial exotoxins or endotoxins, prions, or other similar type living or non- living materials. The term may in particularly refer to cancer cells or cells infected by the polio virus, rhinovirus, piconavirus, influenza virus, or a retrovirus such as the human immunodeficiency virus (HIV).

"Fusion"refers to the joining together of components to form a single contiguous component. For instance, when two cell membranes contact each other the lipids, proteins or other cellular materials re-associate and/or reorganize to form a single contiguous membrane.

"Genetic material"or"therapeutic charge"refers to nucleotides and polynucleotides, including deoxyribonucleic acids (DNA) and ribonucleic acid (RNA). The genetic material may be made by synthetic chemical methodology, may be naturally occurring, or may be made by commonly known recombinant DNA techniques. The nucleotides, DNA, and RNA may contain one or more modified bases or base pairs, or unnatural nucleotides or biomolecules.

"Incorporate"refers to one or more processes for taking up a component, agent, material, cell membrane or biomolecule. Incorporation processes may include invagination, phagocytosis, endocytosis, exocytosis or fusion processes. These processes may or may not further include one or more clathrate coated pits or receptors.

"Intracellular"or"intracellularly"refers to the area within the plasma membrane of a cell, including the protoplasm, cytoplasm and/or nucleoplasm.

"Intracellular delivery"refers to delivery of a bioactive agent, such as a targeting ligand and/or prodrug or drug, into the area within the plasma membrane of the cell.

"Lipid"refers to a naturally occurring, synthetic or semi-synthetic (i. e. modified natural) compound that is generally amphipathic. The lipids typically comprise a hydrophilic component and a hydrophobic component. Exemplary lipids include, for example, fatty acids, neutral fats, phosphatides, oils, glycolipids, surface active agents (surfactants), aliphatic alcohols, waxes, terpenes and steriods. The phrase semi- synthetic (or modified natural) denotes a natural compound that has been chemically modified in some fashion.

"Liposome"refers to a generally spherical or spheroidal cluster or aggregate of amphipathic compounds, including lipid compounds, typically in the form of one or more concentric layers, for example bilayers. They may also be referred to as lipid vesicles or encapsulation vesicles. The liposome may be fonnulated, for example, from ionic lipids and/or non-ionic lipids. Liposomes formulated from non-ionic lipids may be referred to as niosomes.

"Nanoerythrosome"refers to a vesicle structure that is derived from erythrocytes and substantially free of hemoglobin. These vesicles have a size of less than about 1 micrometer to about 0.1 micrometer and are substantially spherical or spheroidal. The term refers to any bioactive agent carrier described in United States Patent No.

5,653, 999 and associated patents or patent applications (herein incorporated by reference in their entirety).

"Nanocomposites"refers to composite structures whose characteristic dimensions are found on the nanoscale. An example is the suspension of carbon nanotubes in a soft plastic host.

"Nanodot"refers to nanoparticles that consist of homogenous material, especially those that are almost spherical or cubical in shape.

"Nanoparticle"refers to any material that can be made, ground or produced on the nanoscale.

"Nanopore"refers to a pore or passage through the structure that has a nanoscale inner diameter, where the inner diameter ranges, in many embodiments from about 0.1 to about 400 nanometers, such as from 10 to 30 nanometers, or from 5 to 10 nanometers.

"Nanorod"or"nanotube"refers to nanostructures that are shaped like long sticks or dowels, with a diameter in the nanoscale and a length not very much longer.

"Nanoscale"refers to phenomena that occur on the length scale between 1 and 100 nanometers.

"Nanostructure"refers to structures whose characteristic variation in design length is on the nanoscale.

"Nanowire"refers to nanorods that can conduct electricity.

"Patient"refers to animals, including mammals, preferably humans.

"Polymer"refers to molecules formed from chemical union of two or more repeating units. Accordingly, included within the term"polymer"may be, for example, dimers, trimers and oligomers. The polymer may be synthetic, naturally occurring or semi- synthetic. The term may refer to molecules that comprise 10 or more repeating units.

"Protein"refers to molecules comprising essentially alpha-amino acids in peptide linkages. Included within the term"protein"are globular proteins such as albumins, globulins and histones, fibrous proteins such as collagens, elastins and keratins. Also included within the term are compound proteins, wherein a protein molecule is united

with a non-protein molecule, such as nucleoproteins, mucoproteins, lipoproteins and metalloproteins. The proteins may be naturally occurring, synthetic or semi-synthetic.

"Receptor"refers to a molecular structure within a cell or on the surface of a cell that is generally characterized by the selective binding of a specific substance. Exemplary receptors include cell surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, immunoglobulins and cytoplasmic receptors for steroid hormones.

"Region of a patient"refers to a particular area or portion of the patient and in some instances to regions throughout the entire patient. Examples of such regions include the eye, gastrointestinal regions, cardiovascular regions (including myocardial tissue), circulatory system, bladder, mucosa, renal region, vascular tissues, as well as disease tissue such as cancerous tissue including prostate, breast, gallbladder, and liver. The term includes, for example, areas to be targeted by a drug delivery device or a bioactive agent. The term refers to both topical and internal organs and tissues. The phrase"vascular"or"vasculature"denotes blood vessels (including arteries, veins, and the like). The phrase"gastrointestinal region"includes the region defined by the esophagus, stomach, small intestine, large intestine, and rectum. The phrase"renal region"denotes the region defined by the kidney and the vasculature that leads directly to and from the kidney and includes the abdominal aorta.

"Region to be targeted"or"targeted region"refers to a region where delivery of a therapeutic is desired.

"Self-assembly"or"self-assembled"refers to activation agents of the present invention that are comprised of individual component parts that self assemble into a larger construct or assembly. The activation agent must be able to self assemble in solution. When assembled, the agent must also be capable of association with a vesicle membrane and capable of altering the permeability of the vesicle membrane upon application of the condition. The activation agent may have both a hydrophobic region and a hydrophyllic region for stable incorporation into a vesicle membrane that is amphipathic.

"Solid-state"or"solid state material"refers to materials that are not biological, biologically based or biological in origin. Such materials may include organic chemicals, synthetic fibers or materials, polymers, plastics, semiconductor materials, silica or silicon based substrates or materials, carbon based materials, quantum dots, artificial bone cylinders, magnetic nanoparticles, nanocrystals, suicide inhibitors, nanodots, nanotubes, nanostructures, or nanowires. These structures may be inserted into, comprise a portion of or be attached to the encapsulation vesicles or activation agents. In certain instances they may also comprise the activation agent. These materials should be capable of activation by an activation condition.

"Suicide inhibitor"refers to synthetic molecules that, upon reacting with an enzyme, produce a product that binds to the enzyme and, therefore, causes the enzyme not to function (to commit functional suicide).

"Surface"or"on the surface"of the encapsulation vesicle refers to being covalently or noncovalently attached to the exterior, associated with the exterior, embedded or partially embedded or forming a pore or channel through. For instance a activation agent on the surface of an encapsulation vesicle may be covalently or noncovalently attached to the exterior of the encapsulation vesicle, it may be embedded or partially embedded in the encapsulation vesicle, or it may create a channel or pore through the encapsulation vesicle. Channels or pores may allow for release of bioactive agents.

Activation agents on the surface of an encapsulation vesicle may be capable of activation by an activation condition.

"Targeting ligand"or"target ligand"refers to any material or substance that may promote targeting of tissues and/or receptors in vivo or in vitro with the therapeutic compositions of the present invention. The targeting ligand may be synthetic, semi- synthetic, or naturally occurring. Materials or substances which may serve as targeting ligands include, for example, proteins, including antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, vitamins, steriods, steriod analogs, hormones, cofactors, bioactive agents, genetic material, including nucleotides, nucleosides, nucleotide acid constructs and polynucleotides.

"Therapeutic"refers to any pharmaceutical, drug or prophylactic agent that may be used in the treatment (including the prevention, diagnosis, alleviation, or cure) of a malady, affliction, disease or injury to a patient. Therapeutic includes contrast agents and dyes for visualization, Therapeutically useful peptides, polypeptides and polynucleotides may be included within the meaning of the term pharmaceutical or drug.

"Tissue"refers generally to specialized cells that may perform a particular function.

The term refers to an individual cell or plurality or aggregate of cells, for example, membranes, blood or organs. The term also includes reference to an abnormal cell or plurality of abnormal cells. Exemplary tissues include myocardial tissue, including myocardial cells, membranous tissues, including endothelium and epithelium, laminae, connective tissue, including interstitial tissue, and tumors.

"Vesicle"or"encapsulation vesicle"refers to an entity that is generally characterized by the presence of one or more walls or membranes that form one or more internal voids. Vesicles may be formulated, for example, from a stabilizing material such as a lipid, including the various lipids described herein, a proteinaceous material, including the various proteins described herein, and a polymeric material, including the various polymeric materials described herein. As discussed herein, vesicles may also be formulated from carbohydrates, surfactants, and other stabilizing materials, as desired. The lipids, proteins, polymers and/or other vesicle forming stabilizing materials, may be natural, synthetic or semi-synthetic. Vesicles may comprise walls or membranes formulated from lipids. The walls or membranes may be concentric or otherwise. The stabilizing compounds may be in the form of one or more monolayers or bilayers. In the case of more than one monlayer or bilayer, the monolayers or bilayers may be concentric. Stabilizing compounds may be used to form a unilamellar vesicle (comprised of one monolayer or bilayer), an oligolamellar vesicle (comprised of more than about three monolayers or bilayers). The walls or membranes of vesicles may be substantially solid (uniform), or referred to as, for example, liposomes, lipospheres, nanoliposomes, particles, micelles, bubbles, microbubbles, microspheres, nanospheres, nanostructures, microballoons, microcapsules, aerogels, clathrate bound vesicles, hexagonal/cubic/hexagonal II phase structures, and the like. The internal

void of the vesicle may be filled with a wide variety of materials including, for example, water, oil, gases, gaseous precursors, liquids, fluorinated compounds or liquids, liquid perfluorocarbons, liquid perfluoroethers, therapeutics, bioactive agents, if desired, and/or other materials. The vesicles may also comprise a targeting ligand if desired.

"Vesicle stability"refers to the ability of vesicles to retain the gas, gaseous precursor and/or other bioactive agents entrapped therein after being exposed, for about one minute, to a pressure of about 100 millimeters (mm) of mercury (Hg). Vesicle stability is measure in percent (%), this being the fraction of the amount of gas which is originally trapped in the vesicle and which is retained after release of the pressure.

Vesicle stability also includes"vesicle resilience"which is the ability of a vesicle to return to its original size after release of the pressure.

Where a range of values is provided, it is understood that each intervening value, to the tenth of a unit of the lower limit unless the context clearly dictates otherwise, between the upper and the lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limit ranges including either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. If for some reasons the usage or definitions herein shall be interpreted to differ from the commonly understand usage, then the definitions, herein, shall prevail. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the methods, devices and materials are now described. Methods recited herein may be carried out in any order of the recited events that are logically possible, as well as the recited order of events.

All patents and other references cited in this application infra and supra, are hereby incorporated by reference except as they may conflict with those of the present application (in which case the present application prevails).

In further describing the present invention, the therapeutic composition components and methods of making the composition are first described in general detail. Then a few representative applications are provided. Subsequently, the method of therapeutic treatment using the therapeutic composition is then described and examples provided.

Activation Agents: An important component of the invention is the activation agent. The activation agent has a number of important properties. For instance, the activation agent is capable of being activated by an activation condition. The activation agent may also be capable of destroying or disrupting the cellular biochemistry of the cell or cell membrane it is in or becomes incorporated into. Activation agents may be capable of being transferred or incorporated into the cell membranes or cellular interior of other cells.

They may also have the capability of destroying or disrupting nearby or adjacent cells.

The activation agent may also be capable of being used as a transmembrane channel to regulate or deliver a drug. The activation agent may be gated to open and close by an activation condition.

Another important property of the activation agent is the fact that it is capable of self assembly in or on the encapsulation vesicles before they are used in vitro or in vivo.

Typically, the activation agent is already in place on the surface of the encapsulation vesicles when administered in vivo. However, the description of self assembly includes those embodiments where the agent is present in a pre-assembled form, but is capable of self assembly prior to or after incorporation into the vesicle. In certain cases, the activation agent has lytic activity. The activation agents may for instance comprise a zeolite, a nanotube, a nanorod, a nanocomposite, a nanowire, an ionophore, a nanodot, a quantum dot, a nanostructure, a plastic, a polymer, a synthetic material, silica or silicon materials, artificial bone or bone material, suicide inhibitors and other known materials. For instance, the activation agent may comprise a self- assembled nanowire positioned in the encapsulation vesicle that may be activated by an exogenous or endogenous source. The nanowire may become incorporated into a

cancer cell (by endocytosis, fusion, or phagocytosis) and then be irradiated by an external light source to"burn out"the tumor.

The activation agents may also comprise a pore forming agent.

Pore Forming Agents: One type of activation agent may be a pore forming agent. The pore forming agents have a number of important properties. The pore forming agent may have lytic activity. The pore forming agent may also be capable of activation by the activation condition and can open, close or both. It also may be capable of releasing chemicals or molecules that may prove toxic to a pathogenic cell. Pore forming agents must be capable of being stably associated with the encapsulation vesicle and may reside on the surface of an encapsulation vesicle. hi certain instances, the pore forming agent may be self assembling, however, self assembly is not a requirement of every pore forming agent as long as the agent satisfies the criteria of the activation agents described above. For example, the pore forming agent alters the permeability of the vesicle in response to an activation condition to selectively and controllably release the bioactive agent without substantial degradation or destruction of the vesicle or vesicle population. For instance, the pore forming agents of the present invention may comprise a biomolecule or solid-state material. Biomolecules may also comprise fusion proteins that may be a pore forming agent or may be used in conjunction with a pore forming agent to dock with a cell membrane or receptor on a cell membrane or surface. Pore forming agents may be designed to hold bioactive agents or degrade to release bioactive agents or other materials that may be potentially toxic to a pathogenic cell upon activation by an activation condition.

Biomolecules In certain embodiments of the invention a lytic pore forming agent may be used that is naturally occurring or synthetically made. The pore forming agent can be a molecule or fragment, derivative or analog of such molecules. The pore forming agents may be capable of making one or more lesions or pores in the encapsulation vesicle (s). These pore forming agents may be derived from a variety of bacteria including a-hemolysin, E. coli hemolysin, E. coli colicin, B. thurifzgensis toxin, aerolysin, perfringolysin,

pneumolysin, streptolysin O, and listeriolysin. Eucaryotic pore forming agents capable of lysing cells include defensin, magainin, complement, gramicidin, mellitin, perforin, yeast killer toxin and histolysin. Synthetic organic molecules that are capable of forming a lytic pore in encapsulation vesicles can also be used. Other synthetic pore forming agents described in Regen et al, Biochem. Biophys. Res.

Commun. 159: 566-571, 1989, herein incorporate by reference.

The composition of the invention can also include fragments of naturally occurring or synthetic pore forming agents that exhibit lytic activity. In addition, the invention provides for biologically active and inactive fragments of polypeptides. Biologically active fragments are active if they are capable of forming one or more lesions or pores in synthetic or naturally occurring membrane systems. Inactive fragments are pore forming agents that are capable of being activated or cleaved into activity by some internal or external event, physical activity, or chemical modification.

The biologically active fragments of lytic pore forming agents can be generated by methods know to those skilled in the art such as proteolytic cleavage or recombinant plasmids.

The invention also includes analogs of naturally occurring pore forming agents that may be capable of lysing cells. These analogs may differ from the naturally occurring pore forming agents by amino acid sequence differences or by modifications that do not affect sequence, or both.

Modifications include in vivo or in vitro chemical derivatization of polypeptides, e. g., acetylation, or carboxylation. Also included in the spirit of the invention are modifications of glycosylation and those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing steps.

The invention also includes analogs in which one or more peptide bonds have been removed and replaced with an alternative type of bond or an alternative type of covalent bond such as a"peptide mimetic". These mimetics are well known in the art.

Similarly, the replacement of the L-amino acid residues is a standard way of rendering the polypeptide less sensitive to proteolysis. Also included are blocking groups that

are used at the amino terminal end including: t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxcarbonyl, fluorenyhnethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4 dinitrophenyl.

Although most modifications are designed to make most proteins more resistant to degradation, the present invention also includes modifications that may be used to enhance such modifications or degradations.

Also within the scope of the present invention are naturally or synthetically occurring organic and inorganic molecules that may be combined with proteins or constructs of the present invention to make them less susceptible to immunological attack. For instance, the compound of the present invention may be coupled to molecules such as polyethylene glycol (PEG) or monomethoxy-polyethylene glycol (mPEG).

The invention also comprises modifications that result in an inactive pore forming agent that can be activated by cell associated substances or conditions. Such modifications can include peptides containing enzymatic cleavage sites (lysine and arginine bonds that can be cleaved) or chemically reactive groups that can be photo- activated. Modifications also include peptides that may be modified to optimize solubility properties or to mediate activation by cell-associated substances.

The invention includes peptides and genetic variants both natural and induced.

Induced mutants can be made in a variety of methods known in the art including random mutagenesis or polymerase chain reaction.

The invention also includes the use of organic and non-organic nanotubes. For instance, these molecules may comprise hollow coiled molecules, linear D, L peptides from cylindrical ß or n helices, helices folding of linear oligophenylacetylenes, ring stacking motifs, tubular ensembles from cyclic D, L-a-peptides, microcrystalline peptide nanotubes, self assembling transmembrane ion channels, pore structures from D, L peptides, cystine macrocycles, serinophanes, carbohydrate nanotubes, tubular mesophases from macrocyclic precursors, sector assembly motifs, nanotubes from

block copolymers, folded sheet motifs, and others. For information regarding potential structures, structure design, construction and applicable materials and agents with the present invention, See"Self-Assembling Nanotubes", M. R. Ghadiri et al., Angew. C111em. Int. Ed. 2001,40, 988-1011 ; WO 95/10535 entitled"Cyclic peptideTube" ; WO 02/090503 A2 and early provisional application entitled "Antimicrobial Peptides and Compositions" (herein incorporated by reference in their entirety).

Solid-State Materials The activation agents may also comprise solid-state materials, especially those that are capable of being on the surface of an encapsulation vesicle. For instance, the activation agent may be or may comprise a zeolite, a nanotube, a nanorod, a nanocomposite, a nanowire, a nanodot, a carbon nanotube, a quantum dot, a nanostructure, a plastic, a synthetic material, silica or silicon materials, artificial bone or bone material, suicide inhibitors and other similar materials known and previously described in the art. Each of these materials is capable of self assembly and activation by an activation condition. Activation may include lytic activity and/or degradation or release of materials that may prove toxic to a pathogenic cell. The activation agent may also comprise a combination or mixture of one or more of these agents.

Targeting Ligand: Targeting ligand refers to any material or substance that may promote targeting of tissues and/or receptors in vivo or in vitro with the compositions of the present invention. The targeting ligand may be optional employed with the present invention.

A key property of the targeting ligand is the ability for the ligand to bind, attach or associate with the surface of a pathogenic cell. The targeting ligand provides the ability to distinguish between healthy and pathogenic cells.

The targeting ligand may be synthetic, semi-synthetic, or naturally occurring.

Materials or substances that may serve as targeting ligands include, for example, proteins, antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, saccharides, including monosaccharides and polysaccharides, carbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors, bioactive agents, genetic material, including

nucleotides, nucleosides, nucleotide acid constructs and polynucleotides. The targeting ligands may include fusion proteins, monoclonal or polyclonal antibodies, Fv fragments, Fab'or (Fab') 2 or any similar reactive immunolgically derived component that may be used for targeting the constructs. Targeting ligands can also include other ligands, hormones, growth hormones, opiod peptides, insulin, epidermal growth factor, insulin like growth factor, tumor necrosis factors, cytokines, fibroblasts or fibroblast growth factors, interleukins, melanocyte stimulating hormone, receptors, viruses, cancer cells, immune cells, B cells, T-cells, CD4 or CD4 soluble fragments, lectins, concavalins, glycoproteins, molecules of hemopoetic origin, integrins and adhesion molecules. Other targeting ligands may be used in conjunction with the photodynamic pore forming agents. For instance, the seringe portion of the diphtheria toxin may be attached to a ligand and the constructs inserted into the encapsulation vesicles. These constructs could then be used to target or deliver the vesicles.

Linkage of Targeting Ligands To Activation Agents and/or Encapsulation Vesicles: The optional targeting ligands may be linked to either or both the activation agents and the encapsulation vesicles by way of covalent or non-covalent bonds. Non- covalent interactions include, but are not limited to ionic, dipole-dipole, van der waals, hydrophobic, hydrophilic, leucine-zipper or antibody-Protein G interactions.

Methods may be used that are well known in the art. It is also within the spirit of the invention, that the targeting ligands may be directly attached to the encapsulation vesicle or to a different protein, activation agent, molecule or biological component embedded in the encapsulation vesicle.

It should also be understood that there are numerous groups that can be used to couple activation agents directly to the encapsulation vesicles. These groups comprise and are not limited to: NH2, COOH, SH and OH groups that are found in abundance in the constituents of the encapsulation vesicles. A variety of important molecules and ligands including antibodies and polyethyleneglycol (PEG) or chemical modifiers such as iminothiolane can be used to conjugate to the membrane surface of the encapsulation vesicle. Linking arms have proven particularly useful in conjugating MPEG's, PEGs or other similar molecules to these membranes. Further modifications of proteins that may be present in the membrane may be accomplished

by using iminothiolane to add SH groups to the membrane or by conjugating the PEG or antibodies using electrophilic groups such as maleimides (Ex: SMCC and the heterobifunctional PEGs). COOH groups or anionic charges can be placed on the encapsulation vesicle by using anhydrides such as succinic, cis aconitic, and citraconic. In contrast, positive charges can be added to the encapsulation vesicle by using polyamines, amines, and amine/amino derivatives that will add-NH2 groups to the surface of the encapsulation vesicle. Numerous type examples are prevalent in the art and include, but are not limited to putrecine, spermidine, and polylysine derivatives. These positive and negative charged membranes are useful for providing additional means for diagnostic separation of bound from unbound encapsulation vesicles.

A number of important frameworks exist for the coupling of MPEG or PEG to encapsulation vesicles to improve the overall immunogenic properties of these vesicles. The following formula has been used as a means for describing the components that are effective for the formation of vesicle-PEG conjugates: Z- (YQ)-X- (CH2-CH2-°) m~ (CH2) n-W-R The"Z"'group is responsible for attaching the PEGs to proteins present in the encapsulation vesicles. This non-exposed group is very important for the overall kinetics of PEG conjugation to the available membrane reactive groups. The reactive groups that the"Z"group could bind or interact with include and are not limited to amino groups such as lysine residues. Two types of reactive groups targeting two families of nucleophiles present on proteins are particularly useful. These groups include NH2 groups (i. e. lysine residues) exemplified by activated esters such as succinimide esters and SH groups (i. e. cysteine residues or iminothiolane derivatives of lysine), exemplified by maleimides, 2-thipyridyl derivatives and disulfide groups.

In one embodiment, "Z"is selected from the group consisting of : COOH, HO, H (aldehyde), OH (hydroxyl) NH2 and SH when YQ = (CH=CH) or

- C (R") =CH2. In addition, if Y =-C= (O)-, then Z = H, N3, OH, CH3,-NH-NH2, an anhydride, mixed anhydride, combinations or an activated ester. Further examples are known to those in that art and described elsewhere.

Numerous methods exist for activating groups for binding MPEG or PEG to encapsulation vesicles and include: cyanogen bromide (BrCN), amino acid ester hydrazine and derivates, oxycarbonlyimidazole derivates, tresylate derivatives, and maleimide derivates.

In order to attach the PEG to the proteins in the encapsulation vesicles, a"YQ" linking arm must also be used. This group is important since there are limited ways for attaching the MPEG or PEG molecules to proteins. In its preferred embodiment, YQ is preferably composed of (CH2) n, where n = 1 to 8 carbons atoms. Greater than 8 carbon atoms tends to render the complex to soluble in the membranes. In addition, it is very difficult to conjugate OH groups of PEGs to proteins. One exception is the cyanuryl chloride derivatives that have proven quite useful in preliminary experiments with encapsulation vesicles. YQ is a member selected from the group consisting of : CH=CH,-C (R") CH2 (where R'= lower alkyl of 1 to 5 carbon atoms), cyanuric chloride, cynaogen halides (Br or Cl) and other OH activating agents such as tosyl and mesyl groups. If Y is a lower alkyl [ (CH2)" ; n=1-7)] ; then Q =-C (O), N, and S.

The"X"atom that connects the MPEG or PEG molecule to the Z- (YQ) portion of the molecule is selected from the group consisting of: sulfur (thioether), oxygen (ether),- N-C (O) (amide), -S-C (O) (thioester),-O-C (O) (ester).

The (CH2-CH2-O) mmoiety of the molecule is the PEG itself and m could be anything from between 1 and 500. PEGs with molecular weights of 350 to 10,000 may be used.

Preferably the PEGs will be in the range of 2,000-5, 000 molecular weight range.

Lastly, the [ n-W-R] group that faces the cytosol or media surrounding the encapsulation vesicle must be considered. This group is particularly important to the present invention.

For example, (CH2) n-W-R should be an inert group such as OCH3 (WR) in order to avoid immune responses in the host or in therapeutic applications. However, in other applications the [ n-W-R] group may be electropositively (-NHa) or electronegatively (COOH) charged. Most applicable to the present invention, [(CH2) n-W-R] group could be SH, or 2-thiopyridyl or maleimide that could be used for either attaching the targeting agent or conjugating all or part of the activation agents to the encapsulation vesicles.

In the formula [ n-W-R], n is = 1 to 7 carbon atoms. In addition, W is selected from the group consisting of: O, N, S, and-C (=O). In addition, if=-0-, R is a member selected from the group consisting of: lower alkyl, H, or cyloalkyl of 1 to 7 atoms or-C (=O)-R, where R is a polyamine, polyamine derivative, spermine, spermine derivatives or putrecine.

If W=-N-, the R is a member selected from the group consisting of : H, lower alkyl, or cycloalkyl of 1 to 7 carbon atoms,-C (=O)- or-C (=O)-R2 where R2 is selected from the group consisting of: spermine, spermidine, putrecine, or a lower alkyl chain of 1 to 6 carbon atoms having one or more P04, S03H or COOH group or groups.

If W =-S-, R is a member selected from the group consisting of : lower alkyl, cycloalkyl of 1 to 7 carbon atoms,-C (=O)-,-C (=O)-R and H, where R is a polyamine deriving from spermine, putrecine, or spermidine.

If W =-C (=O)-, R is a member selected from the group consisting of : lower alkyl, cycloalkyl of 1 to 7 carbon atoms,-C (=O)-,-C (=O)-R and H, where R is a polyamine deriving from spermine, putrecine, or spermidine.

WR is a member selected from the group consisting of : COOH, P04, and S03H.

In addition, it should be mentioned the correct amount of substitution of PEG or other ligands is important to maintain the integrity of the encapsulation vesicles. If substitution is too high it is likely to cause the encapsulation vesicles to collapse.

Approximately, 2-30% of the reactive groups of the membrane of the encapsulation vesicles will be substituted with MPEG or PEG.

The Encapsulation Vesicles: The encapsulation vesicle is important to the present invention and has a few important properties. The encapsulation vesicle must be capable of accommodating the activation agents described herein on its surface, contained within the membrane, or incorporated into the membrane of the vesicle. This may also include the option of being able to attach a targeting ligand to the surface of the encapsulation vesicle. The encapsulation vesicle may also have the ability to encapsulate a bioactive compound.

In addition, the encapsulation vesicle need not be a synthesized material. For instance, it may be naturally occurring or comprise parts of naturally occurring cells. For instance, the encapsulation vesicle may comprise a red blood cell, a white blood cell, a red blood cell ghost, a white blood cell ghost, a pathogenic cell, a diseased cell, or any other cell that has been infected or not infected. However, as discussed above, the encapsulation vesicle must be capable of associating with one or more self-assembled activation agents.

For instance in certain instances"vesicle"or"encapsulation vesicle"refers to an entity that is generally characterized by the presence of one or more walls or membranes that form one or more internal voids. Vesicles may be formulated, for example, from a stabilizing material such as a lipid, including the various lipids described herein, a proteinaceous material, including the various proteins described herein, and a polymeric material including the various polymeric materials described herein. As discussed herein, vesicles may also be formulated from carbohydrates, surfactants, and other stabilizing materials, as desired. The lipids, proteins, polymers and/or other vesicle forming stabilizing materials, may be natural, synthetic or semi- synthetic. Vesicles may comprise walls or membranes formulated from lipids. The walls or membranes may be concentric or otherwise. The stabilizing compounds may be in the form of one or more monolayers or bilayers. In the case of more than one monolayer or bilayer, the monolayers or bilayers may be concentric. Stabilizing compounds may be used to form a unilamellar vesicle (comprised of one monolayer or bilayer), an oligolamellar vesicle (comprised of more than about three monolayers or bilayers). The walls or membranes of vesicles may be substantially solid (uniform), or referred to as, for example, liposomes, lipospheres, MPEG or PEG- coated liposomes, nanoliposomes, nanoerythrosomes, particles, nanoparticles,

micelles, bubbles, microbubbles, microspheres, nanospheres, nanostructures, microballoons, microcapsules, aerogels, clathrate bound vesicles, hexagonal/cubic/hexagonal II phase structures, and the like. The internal void of the vesicle may be filled with a wide variety of materials including, for example, water, oil, gases, gaseous precursors, liquids, fluorinated compounds or liquids, liquid perfluorocarbons, liquid perfluoroethers, therapeutics, bioactive agents, if desired, and/or other materials. The vesicles may also comprise a targeting ligand if desired.

The encapsulation vesicles may also comprise nanoerythrosomes and other lipid based or cellular derived materials. In addition, the vesicles may comprise parts of a cell, other diseased or pathogenic cells capable of fusion or having receptors or fusion proteins on their surfaces. For instance, a potential encapsulation vesicle may comprise a virus such as a T4 phage, an adenovirus, a polio virus, an influenza virus, an HIV virus or other viruses, bacteria, fungi, or pathogenic cells capable of membrane fusion. These vesicles may be naturally occurring or may have been altered physically or chemically through recombinant DNA technology. However, other naturally occurring or non-naturally occurring synthetic and non-synthetic organic or biologically based molecules, polymers and co-polymers are within the scope of the invention. Naturally occurring encapsulation vesicles may comprise erythrocytes, leukocyte, melanocytes, fibroblasts or components of these cells. Other encapsulation vesicles may comprise synthetically designed organic molecules and biodegradable polymers are also within the scope of the present invention.

In other embodiments of the invention, the vesicle may comprise a solid, substantially solid, gel, sol-gel, composite, nanocomposite, nanostructure, nanoporous material, porous nanostructure, nanoshell, nanocrystal, degradable polymer, biodegradable polymer, or device as taught in United States Patent No. 3,948, 254 (herein incorporated by reference). Other structures well known in may include nanostructures that self-assemble. For instance such structures are described by Whitesides et al., Science (1991) 254: 1312-1319. Bates, Science (1991) 251: 898- <BR> <BR> 905; Gunther &amp; Stupp, Langmuir (2001) 17: 6530-6539; Hulteen et al. , J. Am. Chem.<BR> <P>Soc. (1998) 120: 6603-6604; Moore and Stupp. , J. Am. Chem. Soc. (1992): 9-14; Muthukumar et al, Science (1997) 277: 1225-1232; Stupp et al., Science (1997) 276: 384-389 ; Stupp et al. , Science (1993) 259: 59-63; and Zubarev et al. , Science (1999) 283: 523-526.

Activation Conditions or Substances: An important component of the invention is the activation conditions that activate the activation agents. For instance, an activation agent such as a pore forming agent may be activated at the surface of the target cell by conditions or substances that are endogenously provided by the system or target cell or exogenously provided by a source other than the target cell. Physical, chemical or biochemical conditions may be used to activate the lytic activity. Physical conditions include heat, light or temperature changes. Chemical activators include changes in pH or reduction potential, metal ions or protecting groups that may be activated or de-activated. Light sources may include lasers, red lasers, ultraviolet lights, and other optical materials or substances well known in the art. Light wavelengths may include and not be limited to > 300 nm, 400 mn, 500-550 nm, 630-650 nm etc..

In one embodiment of the invention, a removable photoactivatable protecting group may be employed. The pore forming agent or protein becomes inactive by addition of the protecting group. Upon irradiation by an external light or UV source the protecting group is removed and the pore forming agent becomes activated to form pores. Lytic pore forming activity can also be activated biochemically by any substance secreted by a pathogenic cell. These biochemical activators include: proteases, esterases, glycosidase, ectokinases, phosphatases and similar type substance or parts of these substances.

Assembly of the System: The composition of the present invention or components such as the activation agents or pore forming agents, are self-assembling. The composition may be assembled in any order. The composition may be self-assembling or may be assembled manually in a step-wise fashion. It is important to the invention that the composition or activation agents be associated with the encapsulation vesicles before they are used in vivo or in vitro. This insures that they will then be capable of activation by the activation conditions. Self-assembly may be molecular based where there is a spontaneous association of molecules under equilibrium conditions that form stable, structurally well defined aggregates joined by covalent or non-covalent bonds.

Covalent, ionic, dipole-dipole or noncovalent bonds may also be used to attach pore

forming agents to encapsulation vesicles. As noted above, pore forming agents may be positioned on the exterior or may be biomolecules in monomeric or oligomeric forms embedded in the encapsulation vesicles. Components need not be spatially close together, but may be capable of self-assembly upon an endogenous or exogenous condition, chemical or biochemical reaction or response.

Therapeutic Composition: The therapeutic composition comprises an encapsulation vesicle for encapsulating a bioactive agent and an activation agent in the encapsulation vesicle.

FIG. 1 shows a schematic representation of a typical embodiment of the present invention. The figure shows an encapsulation vesicle such as a PEG-coated liposome and an activation agent such as a transmembrane nanotube in the membrane of the encapsulation vesicle. In the figure the activation agent may comprise a D, L-a- peptide or similar type molecule. The nanotube may be designed to be activatable by an activation condition. In certain embodiments the activation agent may be photoswitchable.

FIG. 1A shows an enlarged portion of FIG. 1 with the activation agents as an intramolecular pore. The intramolecular pore may be designed to be gated and may be positioned anywhere in or on the encapsulation vesicle. In the figure the intramolecular pore is transmembrane and may be employed to open and deliver an enclosed bioactive agent to the surrounding medium. In certain embodiments not shown and discussed the intramolecular pore may assume other orientations and positions in the membrane.

FIG. 1B shows a second enlarged portion of FIG1. However, in this embodiment, the activation agent is shown as a combination of an intramolecular pore and a barrel stave. The barrel stave comprises one or more intramolecular pores that group together. In the figure, six intramolecular pore molecules comprise the final barrel stave structure.

FIG. 2 shows a similar therapeutic composition as described in FIGS1, 1A, 1B, but one or more additional lipid bilayers have been constructed in the encapsulation vesicle. This embodiment has the advantage of removing issues of potential leakage from the compositions. For instance, if the nanotubes are incorrectly positioned in the membrane or capped and gated incorrectly there may be a tendency to leak the enclosed bioactive agents. This could present a problem due to loss of drug and inappropriate delivery. By adding and additional encapsulation layer this issue is removed. Theoretically, phospholipases could partially digest or degrade the therapeutic composition while it is in place. Once the outside coating has been removed. The physician can then photoactivate the therapeutic compositions to deliver the drugs or bioactive agents. The invention should not be interpreted to be limited to one additional bilayer only. Other bilayers, monolayers, and multiple layers may be employed.

FIG. 3 shows the therapeutic composition and how it may be employed. The therapeutic composition circulates in the blood stream and is extravasated by tumor tissues. Once in place they are believed to be generally degraded by phospholipases.

In this case, the therapeutic compositions can be photoactivated to release the drug to the area immediately surrounding the therapeutic composition. The drug delivery can be regulated by the size, length, shape and width of the activation agent and pore.

Therapeutic Administration: The composition can be administered to an animal or human suffering from a medical disorder or disease. The composition may be used alone or in combination with other chemotherapeutic or cytotoxic agents. The encapsulation vesicles can contain a bioactive agent used to treat a disease. For example, an oligomeric antisense DNA or therapeutic charge could be used in the carrier for delivery to a diseased or pathogenic cell. Other bioactive agents used for treating cancer and HIV could also be used.

The composition may also be administered by intravenous infusion, subcutaneous injection, or direct injection to the site of infection. The present invention could also be applied topically or aspirated to a tumor site via bronchial passages to treat cancers of the lung. The therapeutic has the unique ability to operate at the cell membrane surface similar to how the immune system operates to destroy diseased cells. The

present invention improves over drugs, prodrugs and bioactive agents since these agents over time may mutate, may become inactivated by diseased cells that are resistant to the drug or bioactive agent.

The present invention also capitalizes on other important interactions with cells. For instance, encapsulation vesicles may be taken up by a diseased cell by a number of mechanisms including contact release, adsorption, fusion, phagocytosis/endocytosis.

In vivo, however, fusion often takes second place to phagocytosis. Under most circumstance, liposomes are cleared far to rapidly from the bloodstream by phagocytic cells for fusion events to occur to any significant event. However materials such as gangliosides, Sendai virus fusion proteins (active fusogenic <BR> <BR> reconstituted Sendai envelopes (RESVs) ), lysolecithin, phosphatidyl ethanolamine, oleic acid, positively charged lipids, detergents and surfactants may be used to increase the rate of the fusion process. More favorable setting for fusion than the reticular endothelial system (RES) would include such areas as the aqueous humor of the eye, cerebrospinal fluid, or following passive absorption to the walls of capillaries (See New, R. R. C. Liposome: A Practical Approach, Oxford University Press, 1997: Chapter 2,85-90 ; Chapter 6,221-239). The present invention has the capability of working with all form of incorporation including but not limited to receptor mediated endocytosis, endocytosis, phagocytosis and pinocytosis. In this case, the pore forming agent can be photodynamically activated and will help speed up the release of the encapsulated contents into the endosomes.

Fig. 4 shows a diagram of how the therapeutic may operate to destroy drug resistant cells. Although the figure shows a particular embodiment of the invention, the drawing is provided for illustrative purpose only. The scope of the present invention should not be construed to be limited to this particular embodiment. Other broad embodiments, applications and components of the invention are illustrated and provided throughout the disclosure.

An encapsulation vesicle such as for example a nanoerythrosome with embedded photo-activatable pore forming agent (1) and attached targeting ligands is used to recognize a specific tumor antigen. The construct is retained by the cancer cell and the therapeutic agent is transferred into the cell cytoplasm (2) via a fusion mechanism

or absorption process. The therapeutic then interferes with the cancer cell's functions and destroys the cell (3 and 4). The inactivated activation agent with attached protecting group is incorporated into the cancer cell's membrane and may be photo- activated upon irradiating by an external light source. The photo-generated pore forming agents are used to guarantee destruction of any therapeutically resistant cancer cells.

FIG. 5 shows a second method of the present invention. In this method of the invention, an encapsulation vesicle comprising an activation agent such as a nanotube and a fusion protein such as the seringe portion of the diphtheria toxin. The components are combined to make the composition. The composition contacts the cell membrane of a diseased cell and fuses with the diseased cell membranes by way of the fusion protein. This promotes the membranes to fuse and to deliver the contents of the encapsulation vesicle to the interior of the diseased cell. In addition, the activation agents such as a nanotube are also delivered to the membrane of the diseased cell. A physician may then photoactivate the activation agent to destroy any remaining resistant diseased cells.

A still further embodiment may include an encapsulation vesicle that contains a bioactive agent. This encapsulation vesicle may be directed to a diseased cell by way of an optional targeting ligand. It may then be activated by the activation condition and the bioactive agent delivered to a prescribed location to destroy localized diseased cells.

Lastly, an encapsulation vesicle may comprise a material such as a nanocrystal or nanoshell that is capable of fusing, binding or attaching to a diseased cell such as a cancer cell. This may be by way of a receptor, binding site, pocket, ligand, bond, charge or other way of association or component. The encapsulation vesicle then delivers the bioactive agent to the diseased cell to destroy it. In this embodiment of the invention the encapsulation vesicle may optionally include the use of an activation agent such as a nanotube. In addition, the encapsulation vesicle, the ligand or the activation agent may be activated.

EXAMPLE 1 Materials Preparation of PEG-coated liposomes is explained in detail. Other encapsulations and vesicles described above may also be employed with the present invention.

Cholesterol (Chol) was obtained from Sigma (St. Louis, Mo). Sphingomyelin (SM), egg phosphatidylcholine (lecithin or PC), partially hydrogenated PC having the composition IV40, IV30, IV20, IV10, and IV1, phosphatidylglycerol (PG), phosphatidylethanolamine (PE), dipalmitoyl-phosphatidyl glycerol (DPPG), dipalmitoyl PC (DPPC), dioleyl PC (DOPC) and distearoyl PC (DSPC) were obtained from Avanti Polar Lipids (Birmingham, Ala). (For details on the preparation of Stealth@ liposomes, See US. Patent No. 5,013, 556, which is herein incorporated by reference in its entirety).

Preparation of PEG-PE Linked by Cyanuric Chloride A. Preparation of activated PEG 2-0-Methoxypolyethylene glycol 1900-4, 6-dichloro-1, 3,5 triazine previously called activated PEG was prepared as described in J. Biol. Chem. , 252: 3582 (1977) with the following modifications. Cyanuric chloride (5 : 5 g; 0.03 mol) was dissolved in 400 ml of anhydrous benzene containing 10 g of anhydrous sodium carbonate, and PEG-1900 (19 g; 0.01 mol) was added and the mixture was stirred overnight at room temperature. The solution was filtered, and 600 ml of petroleum ether (boiling range, 35. degree. -60. degree. ) was added slowly with stirring. The finely divided precipiate was collected on a filter and redissolved in 400 ml of benzene. The precipitation and filtration process was repeated several times until the petroleum ether was free of residual cyanuric chloride as determined by high pressure liquid chromatography on a column (250 X 3.2 mm) of 5-m"LiChrosorb" (E. Merck), developed with hexane, and detected with an ultraviolet detector. Titration of activated PEG-1900 with silver nitrate after overnight hydrolysis in aqueous buffer at pH 10.0, room temperature, gave a value of 1.7 mol of chloride liberated/mol of PEG. TLC analysis of the product was effected with TLC reversed-phase plates obtained from Baker using methanol- water, 4: 1; v/v, as developer and exposure to iodine vapor for visualization. Under these conditions, the starting methoxy polyglycol 1900 appeared at Rf =0. 54 to 0.60.

The activated PEG appeared at Rf=0. 41. Unreacted cyanuric chloride appeared at Rf =0. 88 and was removed.

The activated PEG was analyzed for nitrogen and an appropriate correction was applied in selecting the quantity of reactant to use in further synthetic steps. Thus, when the product contained only 20% of the theoretical amount of nitrogen, the quantity of material used in the next synthetic step was increased by 100/20, or 5-fold.

When the product contained 50% of the theoretical amount of nitrogen, only 100/50 or a 2-fold increase was needed.

B. Preparation of N- (4-Chloro-polyglycol 1900)-1, 3,5-triazinyl egg phosphatidylethanolamine In a screw-capped test tube, 0.74 ml of a 100 mg/ml (0.100 mmole) stock solution of egg phosphatidylethanolamine in chloroform was evaporated to dryness under a stream of nitrogen and was added to the residue of the activated PEG described in section A, in the amount to provide 205 mg (0.100 mmole). To this mixture, 5 ml anhydrous dimethyl fonnamide was added. 27 microliters (0.200 mmole) triethylamine was added to the mixture, and the air was displaced with nitrogen gas.

The mixture was heated overnight in a sand bath maintained at 110° C.

The mixture was then evaporated to dryness under vacuum and a pasty mass of crystalline solid was obtained. This solid was dissolved in 5 ml of a mixture of 4 volumes of acetone and 1 volume of acetic acid. The resulting mixture was placed at the top of a 21 mm. times. 240 mm chromatographic absorption column packed with silica gel (Merck Kieselgel 60,70-230 mesh) which had first been moistened with a solvent composed of acetone acetic acid, 80/20; v/v.

The column chromatography was developed with the same solvent mixture, and separate 20 to 50 ml aliquots of effluent were collected. Each portion of effluent was assayed by TLC on silica gel coated plates, using 2-butanone/acetic acid/water; 40/25/5 ; v/v/v as developer and iodine vapor exposure for visualization. Fractions containing only material of Rf =about 0.79 were combined and evaporated to dryness under vacuum. Drying to constant weight under high vacuum afforded 86 mg (31.2 micromoles) of nearly colorless solid N- (4-chloro-polyglycol 1900)-1, 3,5-triazinyl

egg phosphatidylethanolamine containing phosphorous. The solid compound was taken up in 24 ml of ethanol/-chloroform ; 50/50 chloroform and centrifuged to remove insoluble material. Evaporation of the clarified solution to dryness under vacuum afforded 21 mg (7.62 micromoles) of colorless solid.

EXAMPLE 2 Preparation of the Carbamate-Linked PEG-PE A. Preparation of the imidazole carbamate of polyethylene glycol methyl ether 9.5 grams (5 mmoles) of polyethylene glycol methyl ether obtained from Aldrich Chemical Co. was dissolved in 45 ml benzene that has been dried over molecular sieves. 0.89 grams (5.5 mmoles) of pure carbonyl diimidazole was added. The purity was checked by an infrared spectrum. The air in the reaction vessel was displaced with nitrogen. Vessel was enclosed and heated in a sand bath at 75° C. for 16 hours.

The reaction mixture was cooled and the clear solution formed at room temperature.

The solution was diluted to 50.0 ml with dry benzene and stored in the refrigerator as a 100 micromole/ml stock solution of the imidazole carbamate of PEG ether 1900.

B. Preparation of the phosphatidylethanolamine carbamate of polyethylene glycol methyl ether 10.0 ml (1 mmol) of the 100 mmol/ml stock solution of the imidazole carbamate of polyethylene glycol methyl ether (compound X) was pipetted into a 10 ml pear- shaped flask. The solvent was removed under vacuum. 3.7 ml of a 100 mg/ml solution of egg phospatidyl ethanolamine (V) in chloroform (0.5 mmol) was added. The solvent was evaporated under vacuum. 2 ml of 1,1, 2,2-tetrachloroethylene and 139 microliters (1.0 mmol) of triethylamine VI was added. The vessel was closed and heated in a sand bath maintained at 95 degree C for 6 hours. At this time, thin-layer chromatography was performed with fractions of the above mixture to determine an extent of conjugation on Si02 coated TLC plates, using butanone/acetic acid/water ; 40/5/5 ; v/v/v ; was performed as developer. Vapor visualization revealed that most of the free phosphatidyl ethanolamine of Rf=0. 68, had reacted, and was replaced by a phosphorous-containing lipid at Rf=0. 78 to 0.80.

The solvent from the remaining reaction mixture was evaporated under vacuum. The residue was taken up in 10 ml methylene chloride and placed at the top of a 21 mm times 270 mm chromatographic absorption column packed with Merck Kiesel-gel 60 (70-230 mesh silica gel), which has been first rinsed with methylene chloride. The mixture was passed through the column, in sequence, using the following solvents.

TABLE 1 Volume % of Volume % Methanol ml Methylene Chloride With 2% Acetic Acid 100 100% 0% 200 95% 5% 200 90% 10% 200 85% 15% 200 60% 40% 50 ml portions of effluent were collected and each portion was assayed by TLC on Si02-coated plates, using 12 vapor absorption for visualization after development with chloroform/methanol/water/concentrated ammonium hydroxide; 130/70/8/0. 5%; v/v/v/v. Most of the phosphates were found in fractions 11, 12,13 and 14.

These fractions were combined, evaporated to dryness under vacuum and dried in high vacuum to constant weight. They yielded 669 mg of colorless wax of phosphatidyl ethanolamine carbamate of polyethylene glycol methyl ether. This represented 263 micromoles and a yield of 52.6% based on the phosphatidyl ethanolamine.

An NMR spectrum of the product dissolved in deutero--chloroform showed peaks corresponding to the spectrum for egg PE, together with a strong singlet due to the methylene groups of the ethylene oxide chain at Delta=3. 4 ppm. The ratio of methylene protons from the ethylene oxide to the terminal methyl protons of the PE acyl groups was large enough to confirm a molecular weight of about 2000 for the polyethylene oxide portion of the molecule of the desired product polyethylene glycol conjugated phosphatidyethanolamine carbamate, M. W. 2,654.

EXAMPLE3 Preparation of Ethylene-Linked PEG-PE A. Preparation of I-trimethylsilyloxy-polyethylene glycol 15.0 gm (10 mmoles) of polyethylene glycol) M. Wt. 1500, (Aldrich Chemical) was dissolved in 80 ml benzene. 1.40 ml (11 mmoles) of chlorotrimethyl silane (Aldrich Chemical Co. ) and 1.53 ml (1 mmoles) of triethylamine was added. The mixture was stirred at room temperature under an inert atmosphere for 5 hours.

The mixture was filtered with suction to separate crystals of triethylammonium chloride and the crystals were washed with 5 ml benzene. Filtrate and benzene wash liquids were combined. This solution was evaporated to dryness under vacuum to provide 15.83 grams of colorless oil that solidified on standing.

TLC of the product on Si-Cl8 reversed-phase plates using a mixture of 4 volumes of ethanol with 1 volume of water as developer, and iodine vapor visualization, revealed that all the polyglycol 1500 (Rf=0. 93) has been consumed, and was replaced by a material of Rf =0. 82. An infrared spectrum revealed absorption peaks characteristic only of polyglycols. Yield of I-trimethylsilyoxypolyethylene glycol, M. W. 1500 was nearly quantitative.

B. Preparation of trifluoromethane sulfonyl ester of I-trimethylsilyloxy-polyethylene glycol 15.74 grams (10 mmol) of the crystalline I-trimethylsilyloxy polyethylene glycol obtained above was dissolved in 40 ml anhydrous benzene and cooled in a bath of crushed ice. 1. 53 ml (11 mmol) triethylamine and 1. 85 ml (11 mmol) of trifluoromethanesulfonic anhydride obtained from Aldrich Chemical Co. were added and the mixture was stirred over night under an inert atmosphere until the reaction mixture changed to a brown color.

The solvent was then evaporated under reduced pressure and the residual syrupy paste was diluted to 100.0 ml with methylene chloride. Because of the great reactivity of trifluoromethane sulfonic esters, no further purification of the trifluoromethane sulfonyl ester of I-trimethylsilyloxy polyethylene glycol was done.

C. Preparation of N-1-trimethylsilyloxy polyethylene glycol 10 ml of the methylene chloride stock solution of the trifluoromethane sulfonyl ester of 1-trimethylsilyloxy polyethylene glycol was evaporated to dryness under vacuum to obtain about 1.2 grams of residue (approximately 0.7 mmoles). To this residue, 3.72 ml of a chloroform solution containing 372 mg (0.5 mmoles) egg PE was added.

To the resulting solution, 139 microliters (1.0 mmole) of triethylamine was added and the solvent was evaporated under vacuum. To the obtained residue, 5 ml dry dimethyl formamide and 70 microliters (0.50 mmoles) triethylamine (VI) was added. Air from the reaction vessel was displaced with nitrogen. The vessel was closed and heated in a sand bath at 110 degree C for 22 hours. The solvent was evaporated under vacuum to obtain 1.58 grams of brownish colored oil. A 21 times 260 mm chromatographic absorption column filled with Kieselgel 60 silica 70-230 mesh, was prepared and rinsed with a solvent composed of 40 volumes of butanone, 25 volumes acetic acid and 5 volumes of water. The crude product was dissolved in 3 ml of the same solvent and transferred to the top of the chromatography column. The chromatogram was developed with the same solvent and sequential 30 ml portions of effluent were assayed each by TLC.

The TLC assay system used silica gel coated glass plates, with solvent combination butanone/acetic acid/water; 40/25/5 ; v/v/v. Iodine vapor absorption served for visualization. In this solvent system, the N-1-trimethylsilyloxy polyethylene glycol 1500 PE appeared at Rf=0. 78. Unchanged PE appeared at Rf=0. 68.

The desired N-1-trimethylsilyloxy polyethylene glycol 1500 PE was a chief constituent of the 170-300 ml portions of column effluent. When evaporated to dryness under vacuum these portions afforded 111 mg of pale yellow oil of compound.

D. Preparation of N-polyethylene glycyl phosphatidyl-ethanolamine acetic acid deprotection Once-chromatographed, PE compound was dissolved in 2 ml of tetrahydrofuran. To this, 6 ml acetic acid and 2 ml water was added. The resulting solution was let to stand for 3 days at 23° C. The solvent from the reaction mixture was evaporated under vacuum and dried to constant weight to obtain 75 mg of pale yellow wax. TLC on Si- C18 reversed-phase plates, developed with a mixture of 4 volumes ethanol, 1 volume water, indicated that some free PE and some polyglycol-like material formed during the hydrolysis.

The residue was dissolved in 0.5 ml tetrahydrofuran and diluted with 3 ml of a solution of ethanol water; 80: 20; v: v. The mixture was applied to the top of a 10 mm X 25 mm chromatographic absorption column packed with octadecyl bonded phase silica gel and column was developed with ethanol water 80: 20% by volume, collecting sequential 20 ml portions of effluent. The effluent was assayed by reversed phase TLC. Fractions containing onlyproduct of Rr0. 08 to 0.15 were combined.

This was typically the 20-100 ml portion of effluent. When evaporated to dryness, under vacuum, these portions afforded 33 mg of colorless wax PEG-PE corresponding to a yield of only 3%, based on the starting phosphatidyl ethanolamine.

NMR analysis indicated that the product incorporated both PE residues and polyethylene glycol residues, but that in spite of the favorable-appearing elemental analysis, the chain length of the polyglycol chain has been reduced to about three to four ethylene oxide residues. The product prepared was used for a preparation of PEG-PE liposomes.

E. Preparation of N-Polyethylene glycol by fluoride deprotection.

500 mg of crude N-1-trimethylsilyloxy polyethylene glycol PE was dissolved in 5 ml tetrahydrofuran and 189 mg (0.600 millimoles) of tetrabutyl ammonium fluoride was added and agitated until dissolved. The reactants were let to stand over night at room temperature (20 °C).

The solvent was evaporated under reduced pressure and the residue was dissolved in 10 ml chloroform, washed with two successive 10 ml portions of water, and

centrifuged to separate chloroform and water phases. The chloroform phase was evaporated under vacuum to obtain 390 mg of orange-brown wax, which was determined to be impure N-polyethylene glycol 1500 PE compound.

The wax was re-dissolved in 5 ml chloroform and transferred to the top of a 21 mm X 270 mm column of silica gel moistened with chloroform. The column was developed by passing 100 ml of solvent through the column the following solvents in sequence were used.

TABLE 2 Volume % Volume % Methanol Containing Chloroform 2% Conc. Ammonium Hydroxide/methanol 100% 0% 95% 5% 90% 10% 85% 15% 80% 20% 70% 30% 60% 40% 50% 50% 0% 100% Separated 50 ml fractions of column effluent were saved. The fractions of the column were separated by TLC on Si-C18 reversed-phase plates. TLC plates were developed with 4 volumes of ethanol mixed with 1 volume of water. Visualization was done by exposure to iodine vapor.

Only those fractions containing an iodine-absorbing lipid of about 0.20 were combined and evaporated to dryness under vacuum and dried in high vacuum to constant weight. In this way 94 mg of waxy crystalline solid was obtained of M. W.

2226. The proton NMR spectrum of this material dissolved in deuterochloroform showed the expected peaks due to the phosphatidyl ethanolamine portion of the molecule, together with a few methylene protons attributable to polyethylene glycol.

EXAMPLE 4 Preparation of REVs and MLVs A. Sized REVs (Reverse Phase Evaporation Vesicles) A total of 15 nmoles of the selected lipid components, in the mole ratios indicated in the examples below, were dissolved in chloroform and dried as a thin film by rotary evaporation. This lipid film was dissolved in 1 ml of diethyl ether washed with distilled water. To this lipid solution was added 0.34 ml of an aqueous buffer solution containing 5 mM Tris, 100 mM NaCl, 0.1 mM EDTA, pH 7.4, and the mixture was emulsified by sonication for 1 minute, maintaining the temperature of the solution at or below room temperature. Where the liposomes were prepared to contain encapsulated tyraminyl-inulin, such was included in the phosphate buffer at a concentration of about 4 u, Ci/ml buffer.

The ether solvent was removed under reduced pressure at room temperature, and the resulting gel was taken up in 0.1 ml of the above buffer, and shaken vigorously. The resulting REV suspension had particle sizes, as determined by microscopic examination, of between about 0.1 to 20 microns, and was composed predominantly of relatively large (greater than 1 micron) vesicles having one or only a few bilayer lamellae.

The liposomes were extruded twice through a polycarbonate filter (Szoka, 1978), having a selected pore size of 0.4 microns or 0.2 microns. Liposomes extruded through the 0.4 micron filter averaged 0.17 +. (0.05) micron diameters, and through the 0.2 micron filter, 0.16 (0.05) micron diameters. Non-encapsulated [1251] tyraminyl-inulin was removed by passing the extruded liposomes through Sephadex G-50 (Pharmacia).

B. Sized MLVs Multilamellar vesicle (MLV) liposomes were prepared according to standard procedures by dissolving a mixture of lipids in an organic solvent containing primarily CHC13 and drying the lipids as a thin film by rotation under reduced pressure. In some cases a radioactive label for the lipid phase was added to the lipid solution before drying. The lipid film was hydrated by addition of the desired aqueous

phase and 3 mm glass beads followed by agitation with a vortex and shaking above the phase transition temperature of the phospholipid component for at least 1 hour. In some cases a radioactive label for the aqueous phase was included in the buffer. In some cases the hydrated lipid was repeatedly frozen and thawed three times to provide for ease of the following extrusion step.

The size of the liposome samples was controlled by extrusion through defined pore polycarbonate filters using pressurized nitrogen gas. In one procedure, the liposomes were extruded one time through a filter with pores of 0.4 mu and then ten times through a filter with pores of 0.1 mu. In another procedure, the liposomes were extruded three times through a filter with 0.2 mu. pores followed by repeated extrusion with 0.05 mu. pores until the mean diameter of the particles was below 100 nm as determined by DLS. Unencapsulated aqueous components were removed by passing the extruded sample through a gel permeation column separating the liposomes in the void volume from the small molecules in the included volume.

C. Loading 67Ga Into DF-Containing Liposomes The protocol for preparation of DF labeled liposomes as adapted from known procedures (Gabizon). Briefly, liposomes were prepared with the ion chelator desferal mesylate encapsulated in the internal aqueous phase to bind irreversibly transported through the bilayer by hydroxyquinoline (oxine).

D. Dynamic Light Scattering Liposome particle size distribution measurements may be obtained by DLS using a NICOMP Model 200 with a Brookhaven Instruments BI-2030AT autocorrelator attached, operated according to the manufacturer's instructions. The Particle size distribution results are typically expressed as the mean diameter and standard deviation of a Gaussian distribution of vesicles by relative volume.

EXAMPLE 5 Use of a Nanotube as a Carrier: After preparation of lipsomes, self-assembling nanotubes may be added to the composition. In certain embodiments of the invention, the nanotube carriers may be

loaded with a drug molecule before or after self assembly in the liposome or membrane systems. In addition, the nanotube carriers may be added to the system before the MPEG or PEG is added to the exterior of the liposome.

As a preliminary test, the cyclic peptide tubes disclosed may be assembled in the presence of hydrogen peroxide. After tube assembly and particle formation, the mixture is centrifuged to create pellets of the particles containing the nanotubes. The pelleted particles are washed by a further centrifugation step and then combined with the reagents of a bioluminescent assay designed to test for the presence of hydrogen peroxide. Bioluminescence is observed to be confined to the pelleted fraction. This demonstrates that the cyclic peptide tubes can encapsulate hydrogen peroxide within their channel region but that the hydrogen peroxide slowly leaks by diffusion from such channel region into the external media. In the examples shown in FIGS. 1-7 the nanotubes may be optionally loaded with drugs that would be used in drug delivery.

For instance, the nanotube may comprise a small molecule (i. e. a nucleotide, peptide, protein, oligonucleotide, nucleoside, lipid, carbohydrate, monosaccharide, <BR> <BR> disaccharide, RNA, double stranded RNA, DNA, double stranded DNA etc. ) and their derivatives or modified structures). The nanotubes may be employed with the use of a cancer therapeutic such as doxorubicin or daunorubicin or other drugs well known in the art. In addition, the nanotubes may also be assembled into the complex and then loaded with the drug. This has a few advantages. First of all, this provides for the ability to load additional drug material into the overall composition. Secondly, it may serve as a secondary capping function that prevents the leakage of drug material after composition construction. This allows for maximal loading of drugs if an active loading process is used in the construction of the therapeutic composition. In addition, once the liposomes have been degraded, some of the nanotubes may still remain to deliver drug over a prescribed or estimated period of time (based on the size of the tubes and their robustness to degradation by phospholipases). This provides an added advantage for regulation of drug delivered as well as amounts of overall drug that can be stored and delivered to a defined area for needed treatment.

The pore size of the self-assembled organic nanotubes is selectively determined by adjusting the ring size of the peptide sub-unit employed. The internal diameter of the nanotube ensembles can be rigorously controlled simply by adjusting the ring size of

the peptide subunit employed. The This flexibility characteristic can be exploited for improved regulation of drug delivery. A twelve-residue cyclic peptide structure, i. e., the thirty six-membered cyclic peptide subunit cyclo [- (Gln-D-Ala-Glu-D-Ala) 3-1 has been designed and shown to undergo a proton-induced self-assembly process to produce highly ordered nanotubular objects having a uniform 13 A internal van der Waals diameter. These nanotubes have been characterized by IR spectroscopy, low- dose electron microscopy, and the analysis of electron diffraction patterns. The ability to design specifically sized tubular nanostructures is expected to have important applications in catalysis, inclusion chemistry, and molecular electronics.

Formation of the tubular structures is supported by high resolution imaging using cryo electron microscopy, electron diffraction, Fourier-transform infrared spectroscopy, and molecular modeling.

According to the disclosed design principles, cyclic peptide structures that are made up of an even number of alternating D-and L-amino acid residues can adopt a flat ring-shaped conformation in which all backbone amide functionalities lie approximately perpendicular to the plane of the ring structure. In this conformation, the peptide subunits can stack, under favorable conditions, to furnish a contiguous hydrogen bonded hollow tubular structure. The internal diameter of the nanotube ensemble can, in principle, be tailored by adjusting the ring size of the peptide subunit employed. This provides the advantage of being able regulate the prescribed level of drug for delivery. The largest pore diameter peptide based nanotube structure thus far constructed utilizes a thirty six-membered ring peptide subunit cyclo [- (Gln-D-Ala- Glu-D-Ala) 3-1. The requisite peptide subunit was synthesized on a solid-support, <BR> <BR> according to the method of P. Rovero et al. (Tetrahedran Lett. , (1991), vol. 32, pages 2639-2642) and characterized by mass spectrometry and 1H NMR spectroscopy.

Controlled acidification of alkaline solutions of the peptide subunit upon standing afforded rod shaped crystalline materials, as indicated above. Transmission electron microscopy, indicates that each particle is an organized bundle of tightly packed nanotubes. Low dose cryo microscopy, according to the method of M. Adrian et al.

(Nature (1984), vol. 308, pages 32-36) and of R. A. Milligan et al. (Ultramicroscopy (1984), vol. 13, pages 1-10) revealed longitudinal striations with spacing of approximately 25 A as expected for the center to center spacing for closely packed nanotubes. Electron diffraction patterns display axial spacing of 4. 80 A that is in

agreement with the peptide stacking and the formation of tight network of hydrogen bonded (3-sheet type structure. The Meridonial is spacing in the electron diffraction patterns display spacing of 12. 670. 06 A, and 21. 940. 05 A characteristic of a hexagonal body centered packing of nanotubes. Hexagonal lattice resulting from the close packing of cylinders of radius r displays the characteristic two principle lattice planes of radius r and r such as the one observed here (r=12. 67 A and r =21.94 A).

The periodicity in this packing produces diffraction spots at 1/r, 2/r, and so on, and at 1/1, and 2/r, and so on. The observed electron diffraction patterns on the meridional axes extend to third order reflections (4.1 A) signifying the ordered and crystalline state of the nanotube particles. The diffraction patterns also showed a unit cell with an angle of 990 and no other symmetry than the center of symmetry due to Friedel's law. A three-dimensional model of the nanotube structure was built using the parameters obtained from the electron diffraction patterns-unit cell with a=9. 6 A (2x4. 80 A for the antiparallel dimer), lb=c=25.66 A (2x12. 67=Cos9), a=1200, and B==99°. The model shows structure factors similar to the patterns observed in elsewhere in the electron diffraction, thus supporting the proposed three-dimensional model. Involvement: of intermolecular hydrogen bonding network in the tube assembly is also supported by FT-IR spectroscopic analysis according to the method of S. Krimm et al. (Advances in Protein Chemistry; Anfinsen, C. B. , Edsall, J. T.; Richards, F. M. Eds.; Academic Press: Orlando, 1986, pages 181-364). Nanotubes display characteristic IR features of a fi-sheet structure signified not only by the amide I bands at 1626 cm''and 1674 cm~l and an amide 11 band at 1526 cm'\ but also by the observed NH stretching frequency at 3291 cm-1 supporting formation of a tight network of hydrogen bonds. The IR spectrum is very similar to other nanotubes and closely resembles that of crystalline Gramicidin A that is known to form dimeric p- helical structures. Gramicidin A has amide I bands at 1630,1685 W, an amide 11 band at 1539 cm'\ and an NH stretching frequency at 3285 cxri l. Q. M. Naik et al. in Biophys. J. (1986), vol. 49, pages 1147-1154. ) The observed frequency of NH stretching mode correlates to an average intersubunit distance of 4.76 A that is in close agreement with the value of 4.80 A obtained independently from the electron diffraction patterns.

EXAMPLE 6 Ion Channels From Self-Assembling Peptide Nanotubes: Artificial membrane ion channels may be constructed using self-assembled cylindrical peptide architecture. The construct described displays an efficient channel-mediated ion transport activity with rates exceeding 107 ions/sec rivaling that of many naturally occurring counterparts. Such molecular assemblies are expected to have potential utility in the design of novel cytotoxic agents, membrane transport vehicles, and drug delivery systems.

According to the design principles herein disclosed, cyclic peptide structures made up of an even number of alternating D-and L-amino acid residues can adopt a flat ring conformation and stack, under favorable conditions, to furnish a contiguous hydrogen bonded hollow tubular structure. Therefore, an ensemble made up of eight to ten subunits each separated by the expected inter-subunit distance of 4.7 to 5. 0 A and decorated with appropriate hydrophobic surface residues, would be long enough to span the thickness of average biological lipid membranes. The eight residue cyclic peptide cyclo [- (Trp-D-Leu) 3-Gln-D-Leu-) (Sequence No.: 9, See WO 95/10535) was designed for the purpose in hand. It is composed of alternating L-tryptophan and D- leucine side chain moieties with the exception of one L-glutamine residue introduced mainly to simplify the peptide synthesis. It is demonstrated that the channel structures having an aqueous pore of approximately 7.5 A in diameter would form spontaneously upon incorporation of a sufficient concentration of the peptide monomer in lipid bilayers. The driving force for the self-assembly of the channel structure is primarily provided by the enthalpic contribution of a large number of hydrogen bonding interactions which are favored in the low dielectric constant medium of lipid bilayers and by the increase in the lipid chain entropy arising from side chain-lipid interactions. (See D. M Engelman et al. in Cell (1981), vol. 23, pages 411-422; L. C. Allen in Proc. Natl. Acad. Sci. USA (1975), vol. 72, pages 4701-4705; and D. M. Engelman in Annu. Rev. Biophys. Chem. (1986), vol. 15, pages 321-353).

In short, it is demonstrated that the designed flat ring-shaped cyclic peptide is not only structurally predisposed toward intermolecular interaction, but is also energetically favored to self-assemble, in the lipid bilayer environment to furnish the desired transmembrane channel structure.

The following studies using a variety of spectroscopic techniques, lipid vesicle model systems, and single ion channel recordings support the validity of the above design hypothesis. Addition of the peptide subunit to aqueous liposomal suspensions effects a rapid partitioning of the subunit into the lipid bilayers and its spontaneous self- assembly, into ion transport-competent : membrane channel structures. Incorporation of the peptide subunit into lipid bilayers using large unilamellar vesicles has been established by absorption and fluorescence spectroscopy. Formation of the hydrogen- bonded transmembrane channel structure in phosphatidylcholine liposomes has been supported by FT-IR spectroscopy. The observed amide-I band at 1624 cm''is not only similar to the carbonyl stretching frequencies found in other nanotube structures disclosed herein, but is also consistent with the infrared spectrum of gramicidin A in similar lipid bilayers. (E. Nabedryk et al in Biophys. J. (1982), vol. 38, pages 243- 249. Furthermore, the observed N-H stretching frequency at 3272 cm''strongly supports the formation of a tight network of hydrogen bonds with an average intersubunit distance of 4.7 A. Formation of transmembrane channels was also inferred from its highly efficient proton transport activity. Vesicles were prepared having pH 6.5 inside and pH 5.5 in the outside bulk solution. The collapse of the imposed pH gradient in these vesicles, upon formation of the putative transmembrane channel structure, was studied by monitoring the fluorescence intensity of an entrapped pH-sensitive dye. (V. E. Carmichael et al, in J. Am. Chem. Soc. (1989), vol.

111, pages 767-769). Addition of the peptide to such vesicles suspensions causes a rapid collapse of the pH gradient. Unilamellar vesicles were prepared by the reverse- phase evaporation using DPPC, OPPC, cholesterol in the ratio of 1: 1: 2 in a solution containing 5 (6) -carboxyfluorescein (20 Vol in phosphate/saline buffer: 137 WK NaCl, 2.6 mM KC1,6. 4 Vol Na2HP04 1.4 Vol KH2PO4 pH 6.5) according to the method of F. Szoka et al. in Proc. Natl.. Acad. Sci. USA (1978), vol. 75, pages 4194- 4198. Liposomes were then sized by multiple extrusions through Nucleopore polycarbonate membranes (10 times, 50 psi, using 0.8 and 2x 0.4 micron filter stacks) and the untrapped (6)-carboxyfluorescein was removed by size exclusion chromatography (Sephadex G-25 column 1x30 cm) using the same phosphate/saline buffer according to the method of F. Olson et al. in Biochim. Biophys. Acta (1979), vol. 557, pages 9-23. Vesicles formed in this way are approximately 150 nanometer in diameter as determined by electron microscopy. (R. R. C. New, Ed. Liposomes, Oxford university Press, 1990). In each experiment, 70 ml of the stock vesicle

solution (3.5 X 10-3 M in phospholipids) was added to pH 5.5 buffer (1.3 ml, 137 Vol NaCl, 2.6 Vol KC1, 6.4 Na2HP04 1.4 KH2P04) and placed in a 1 cm quartz cuvette inside a stirring thermojacketed sample holder of the florescence instrument and equilibrated at 25° C for 15 minutes with gentle stirring. To the cuvette, through an injector port, 25 ml of the channel forming compounds in DMSO was added with continuous fluorescence monitoring al: 520 nm (excitation at 470 nm). The observed data were then normalized for comparison into the fractional change in fluorescence (Io-It/Io) (V. E. Carmichael et al, in J. Am. Chem. Soc. (1989), vol. 111, pages 767- <BR> <BR> 769) ). According to these experiments, the apparent ion transport activity of cyclo [- (Trp-D-Len) 3-Gln-D-Leu-J] is similar to, if not higher than, that of gramicidin A and amphotericin B. The lipid bilayers used were formed on the tip of patch pipettes using a mixture of synthetic P 0 P E: P 0 P (4: 1) (1-palmitoyl-2-oleoyl-Sn-glycero-3- phosphatidylethanolamine and serine). Five to 10 ml of the peptide solution (1 x 10-7 or 2.0 x 10-6 M in 25% DMSO in buffer solution containing 500 mM NaCl or KC1, 5 mM Cal210 mM HEPES, pH 7.5) was added to 150 ml subphase volume resulting in spontaneous partitioning of the peptide into the membrane.

Ion channels formed spontaneously after peptide was added to the subphase of lipid bilayers. Ion channel activity was observed in 14 out of 22 membranes under symmetrical solutions of 500 mn is NaCI or KC1, 5 mM CaCl2 10 mM HEPES, pH 7.5. Data acquisition and analysis may be performed on a Gateway 2000/486 computer using pCiamp software package and TL-1 labmaster interface. Acquisition rate was 0.1 ms and data were filtered at 2 kHz. Control studies, monitoring the release of 5 (6) carboxyfluorescein dye, indicated that the collapse of the pH gradient was not due to the rupturing of the liposomes nor due to the small amounts of organic solvents (<2% DMSO) employed in these studies. Furthermore, the control peptide [MeGln-D-Leu) 4] which lacks the appropriate surface characteristics for partitioning into the lipid bilayers, but otherwise quite similar in design to the channel forming peptide described above, does not display any ion transport activity under similar conditions. The second control peptide cyclo [MeN-D-Ala-Phe) 4] which has the desirable hydrophobic surface characteristics but lacks the propensity for participating in extended hydrogen bonding network, was also designed and tested for ion transport activity. The peptide design incorporates a novel N-methylation strategy on one face of the ring structure which predisposes the subunit toward a dimeric cylindrical

structure (Ghadiri, M. R. , Kobayashi, K. , Granja, J. R. , Chadha, R. , and McRee, D. E. manuscript in preparation) such a dimeric cylindrical ensemble is approximately 10 A thick and can not span the lipid bilayer. Although the peptide has been shown to partition effectively into lipid bilayers, it does not promote proton transport activity irk the above vesicle experiments. Together, these experiments suggest that not only the hydrophobic surface characteristic of the channel forming peptide is an important factor, but also the peptide subunit must be able to participate in extended hydrogen- bonded stacking interactions to produce channel structures is long enough to span the lipid bilayer.

The designed transmembrane ensemble also shares important characteristics with natural ion channel formers such as gramicidin A and amphotericin B. First, the peptide shows concentration dependence effects on the rate of channel formation (data not shown). Second, when low concentrations of the channel forming peptide is used in the above proton transport experiments, only part of the vesicle population goes to equilibrium very fast. This phenomenon reflects the statistical distribution of the channel forming species among the vesicle population-only part of the population has enough channel-forming molecules to form permeable competent structures in an all-or-none type of a process. Unlike"ion carriers"such as morkensin and valinomycin which bind to metal ions and partition between aqueous-phase and the lipid-phase in order to establish ion equilibrium across the membrane, channel forming species at low concentrations (the designed peptide here, amphotericin B, gramicidin A, and others) because of their inability to defuse back out of the membrane, cannot penetrate other vesicles and, unlike ionophores, cannot easily establish proton or ion equilibrium in all vesicles present in solution. Therefore, the observed rapid proton efflux in the above types of experiments simply reflects the rate limiting step of peptide diffusion into the lipid bilayer and self-assembly into ion- transport competent channel structures and does not reflect the actual rate of channel- mediated ion transport which can occur on a much faster time scale.

EXAMPLE 7 Capped Nanotubes: In another embodiment, a capping structure is provided at the terminus of a self- assembled molecular tube utilized as the activation agent of the invention. It is

evident from Figure 6 and 7 that the subunits at the channel openings, i. e. , at the"cap" positions, are unique with respect to their mode of interaction with the other subunits as well as the micro-environment in which they reside. The peptide subunits at the cap position participate in backbone-backbone hydrogen bonding with only one other subunit and on only one side of the backbone structure. The cap subunits also reside in the amphiphilic micro-environment of the lipid-water interface. The key structural requirements for producing a multiple ring-stacked tubular structure is the spatial disposition of the backbone hydrogen bond donor and acceptor sites on both faces of the peptide ring structure. However, if the cyclic peptide subunit is devoid of hydrogen bond donation from one face of the ring structure through the blocking, e. g., alkylation of backbone amide nitrogen functionalities of one of the chiral moieties present, such a cyclic subunit cannot participate in an extended hydrogen bonding network, but serves to cap a tubular structure. Such selectively alkylated cyclic peptides in non-polar solution are predisposed to dimerization. However, the addition of such selectively alkylated cyclic peptide subunits to the aforementioned ring-stacked tubular structure in an appropriate solvent permits capping or termination of the ring-stacked tubular structures by the monomeric, selectively alkylated cyclic peptides. One such cyclic peptide is shown in FIG. 6 and 7. In this situation, the cyclic peptide has been first alkylated or thioalkylated to form the final capped structure RSH. A number of techniques are possible for introducing the alkylthio group onto the nitrogen cap region of the nanotube (See Protein Function: A Practical Approach, edited by T. E. Creighton, Chapter 10, T. Imoto and H. Yamada, Pages 263-268, Oxford University Press, 1989). Reagents such as 2-mercaptoethanol or dithiothreitol can then be employed to reduce the compound. The complex is then reacted with BNPA (construction discussed below) to form the final CNB-thioester.

The CNB group blocks the channel or pore and drug or encapsulated molecules can not escape. The CNB group can be removed by irradiation at wavelengths (300 nm) that do little damage to most biological tissues. This opens the pore and allows encapsulated drug to be released according to diffusion rates, the length and width of the channel and the concentration of drug in the encapsulation vesicle. R denotes the nanotube. FIG. 6 and FIG. 7 show the photoswitchable nanotube with CNB group attached. The arrows in FIG. 7 show the potential point of photolysis. The full reaction is shown in FIG. 7. The invention should not be interpreted to be limited to this protection group. Other groups well known in the art may be employed.

The most meaningful way of classifying the various amino acids is on the basis of the polarity of their R groups in water near pH 7. There are four main classes: (1) nonpolar or hydrophobic, (2) polar but uncharged, (3) positively charged, (4) negatively charged. The nonpolar or hydrophobic group includes amino acids with aliphatic R-groups, such as alanine, leucine, isoleucine, valine, and proline, amino acids with aromatic ring R-groups, such as phenylalanine and tryptophan, and one amino acid with a sulfur-containing R-group, methionine.

Molecular modeling indicated that methylation of backbone amide nitrogen functionalities at all alanine residues would be sufficient to effectively prevent one face of the putative peptide ring structure from participating in intermolecular hydrogen bonding and ring stacking interactions. The peptide was also designed to have an allowed symmetry for favorable intermolecular packing interactions in the solid-state thus permitting its detailed structural characterization by X-ray crystallographic techniques. Amino acids having hydrophobic R-groups were chosen for this Example so as to make the subunit and the resulting dimer ensemble soluble in nonpolar organic solvents. However, the choice of the R group in any given instance depends on the desired solubility characteristics of the end product. The linear form of the target sequence H2N-(L-Phe-D-MeNAla) 4_CO2H was synthesized according to standard solid-phase methods described herein above and then cyclized in solution to furnish the desired cyclic peptide subunit using the following procedure.

A solution of the linear peptide in DMF (1 mM) was treated with TBTU (2- (1H- <BR> <BR> Benzotriazol-i-yl)-1, 1, 3, 3- tetramethyluronium tetrafluoroborate, 3 mM), HOBt (J. - Hydroxybenzotriazole, 3 MM) and DIEA (diisopropylethylamine, 1% v/v) at YC for 12 h to give the desired cyclic peptidemonomer, after reverse-phase HPLC purification, in 70% yield, which can then be used for capping or terminating peptide nanotubes. The 1H-NMR spectrum of the produced peptide subunit in polar solvents, such as deuterated methanol or dimethyl sulfoxide (DMSO), displays multiple slow- exchanging conformational isomers due to the well-known propensity of secondary amides toward cis-trans isomerization. Variable temperature INTMTR experiments in DMSO indicate cis-trans conformational activation barriers on the order of 16 to 17 kcal-mol4. However, in nonpolar solvents such as carbon tetrachloride (CC14) or deuterochloroformm CDC13 the peptide exists an all trans flat-ring-shaped backbone

conformation that is in dynamic equilibrium with the expected dimeric cylindrical ensemble. The monomeric peptide subunit displays a temperature independent (from 40 to 55° C in CDC13) and highly symmetrical IH NMR spectrum excluding the possibility of an intramolecularly hydrogen bonded conformation. The preponderance of a flat ring-shaped backbone conformation is also indicated by the observed 7.5 Hz JNH-CaH coupling constant. Intermolecular hydrogen bonding interactions producing the stacked dimeric ensemble are signified by the expected downfield shift of the phenylalanine N-H backbone resonance from 6.98 to 8.73 ppm (JNH-CaH =8.5 Hz) and are unequivocally established by the observed exchange and NOE cross peaks in its ROESY spectrum. ROESY experiments were performed on a Bruker AMX-500 with 300 ms spin lock (mixing) time using Bruker's standard pulse program. Data were processed using FELIX software. Time domain data was apodized using skewed sine-bell squared window functions. Zero-filling was used to obtain the final data size of 1024 x 1024 complex matrix. A. Bax D. G. Davis, J. Magnetic Resonance 1985,63, 207-213. Formation of a tight hydrogen bonded ensemble with an average inter subunit N-O distance of 2. 95 A is also evidenced by the appearance of an N-H stretching band in the infrared spectrum at 3309 cm-1. As expected, the self-assembly process displays concentration and solvent dependent spectra with the association constants Ka. (CCl4) =1. 4x104 M-1 and Ka), (CDC13) =1. 26i0. 13 x 103 M-1 at 293 K.

The association constants reported are the lower limits due to the presence of small amounts of included water in the peptide samples. When water is rigorously excluded (4 Ka molecular sieves) the association constant Ka (CDC13) =1260 M-1 is approximately doubled to Ka (CDCl3) =2540 M-1 The Ka (CC14) reported was performed in a mixture of 84% CC14 and 16% CDC13 for solubility reasons. Variable temperature studies (van't Hoff plots) establish the following thermodynamic parameters for the dimerization process in chloroform : A Cp=203.1 cal. K~l. mol'\ A H°29s=11. 0 kcal. mol-1, A S°29S=-23. 7 that clearly supports the expected enthalpic contribution of intermolecular hydrogen bonding interactions (0.5 to 0.7 kcal-mol4 for each hydrogen bonding interaction) as the major driving force in the self-assembly process. The above experiments indicate that the peptide subunit adopts a flat ring- shaped solution conformation that is energetically favored toward ring stacking and intermolecular hydrogen bonding interactions by 4.0 to 5.6 kcal-mol-1, depending on the solvent employed. It follows then that an additive gain in free energy of stabilization is to be expected as the number of ring-stacking interactions are

increased. This is particularly relevant to the self-assembled nanotubes and to the transmembrane ion channel structures that can be produced. Colorless prismatic crystals suitable for X-ray analysis were obtained from the solutions of the peptide in water-saturated dichloromethane by vapor-phase; equilibration with hexane. The crystal structure was solved in the space group I422 with a final R-factor of 8. 87%.

Data were collected on a Rigaku AFC6R diffractometer equipped with a copper rotating anode (CuK) and a highly oriented graphite monochromator. The structure was solved in the space group 1422 with a final R-factor of 8. 87% and weighted R- factor of 10.35% and the residual electron density having unique reflections with 4a (F). The unit cell parameters are a = b= 16.78, and a c=21.97 A. The solid-state structure is a cylindrical is dimeric ensemble, analogous to the solution structure deduced from the 1H NMR and FT-IR analyses, corroborating very well the previously calculated nanotube structures derived primarily from the analysis of electron diffraction patterns. The dimeric ensemble is a combination of a flat ring- shaped cyclic peptide subunit with backbone amide groups perpendicular to the plane of the ring structure and at crystallographic four-fold rotation axis parallel to the c axis passing through the center of the peptide ring. Two peptide subunits are closely stacked in an antiparallel orientation and are related by a two fold rotation along either a or b axis. The b-sheet-like cylindrical ensemble is stabilized by eight intersubunit hydrogen bonding interactions with an inter subunit N-0 distance of 2. 90 A. It is noteworthy that the distance of 2.95 A inferred from the observed NH stretching band at 3312 CM-1 in the FT-IR spectrum is remarkably consistent with the crystallographic; measurements. The cylindrical ensemble has an approximate 7.5 A van der Waals internal diameter and a 450 A3 volume. The tubular cavity is filled with partially disordered water molecules, establishing the hydrophilic internal characteristics of the peptide nanotube structures. The ensemble packs in the crystal in a body centered fashion to produce a continuously channeled super-lattice structure along the c axis. The interior surface characteristics of the channels alternate approximately every 11 A between the hydrophobic domains, created by the aromatic phenyl moieties, and the hydrophilic interior of the peptide cylindrical ensemble.

Water molecules near the hydrophobic domains are considerably more disordered, displaying only a weak residual electron density. The observed water electron density is the time average of water molecules binding at: multiple; overlapping sites; suggesting a facile movement of loosely held water molecules within the cavity. This

observation that can be attributed to the lack of a discrete, strong binding site (s), is an important attribute of the produced cyclic peptide structures and is believed to contribute to the remarkable transport efficiencies of the formed transmembrane ion channels. The foregoing discussion and the accompanying examples are presented as illustrative, and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.

EXAMPLE 8 Gated Nanotubes Cyclic peptide tubes can also be employed as ion-gated membrane channel structures.

By the appropriate choice of the amino acid side chain moieties one can tune, at will, the surface characteristics of the self-assembled cyclic peptide tubes. For the purpose of constructing membrane channel structures, cyclic peptides are designed to have hydrophobic side chain moieties in order to ensure their insertion and self-assembly within the nonpolar environment of lipid bilayer membranes. The anatomy of the tubular membrane channel structure is schematically shown elsewhere. It consists of approximately eight stacks of anti-parallel peptide subunits which enables the channel structure to span the thickness of the average biological lipid membrane-according to our previous electron diffraction studies on the self-assembled organic nanotubes, an average intersubunit distance of 4.8 to 5.0 A is expected for such an extensively hydrogen bonded antiparallel p-sheet ensemble. The channel structure can form spontaneously upon the dissolution of a sufficient concentration of the peptide monomer in the lipid bilayer. The driving force for the self-assembly of the channel structure is provided by a) the enthalpic contribution of a large number of highly favorable and oriented hydrogen bonding interactions-each hydrogen bond in the nonpolar environment of the membrane is estimated to worth about 5-6 kcal. mol-1 (for a channel composed of eight 8-mer cyclic peptides, the hydrogen bond network consists of 56 highly cooperative intermolecular hydrogen bonds), and b) by the increase in the lipid bilayer entropy arising from the side chain-lipid interactions.

These highly favorable energetic contributions easily compensate for the loss of entropy associated with the peptide self-assembly and self-organization. Furthermore, considering that only hydrophobic residues are utilized in the peptide design, the unfavorable backbone dissolvation energy does not play a significant role in the

assembly process especially since the hydrophilic interior of the channel structure is expected to be filled with a large number of interacting water molecules. In short, such de novo designed cyclic peptides are not only structurally predisposed toward intermolecular interaction, but are also energetically favored to self-assemble, in the lipid bilayer environment, into artificial membrane channels. Furthermore, if needed, simple methods are available for linking the subunits together through side chain-side chain covalent bond formation in order to obtain a permanently fused molecular channel structure. The self-assembled channels have two important and unique structural features that are pertinent hereto. One feature is that the channel pore size can be easily tailored by choosing an appropriate ring size for the cyclic peptide subunit. This allows for the design of shape-selective membrane pore structures. The second feature produces channel gating--the process by which molecular transport across the channel is turned on or off. It is evident that the two subunits at the channel entrance, i. e. , the"cap"position, are unique with respect to their mode of interaction with the other subunits as well as the micro-environment in which they reside. The peptide subunits at the cap position participate in backbone-backbone hydrogen bonding with only one other subunit and on only one side of the backbone structure.

The cap subunits also reside in the amphiphilic micro-environment of the lipid-water interface. These unique characteristics can be exploited for the design of gated membrane channels in the following fashion. In order to ensure segregation of the cap subunits from the other channel forming subunits, one face of the backbone structure can be blocked from participating in inter-subunit hydrogen bonding interactions simply by alkylating the backbone amide nitrogen functionalities at the homochiral residues. Such N-alkylated species not only lack hydrogen bonding donor capability on one face of the disk structure but also severe steric interaction imposed by the N- alkyl substituents effectively prevents the participation of the peptide subunit in bi- direction hydrogen bonding stacking interactions. Therefore, such N-alkylated subunits can only reside at the cap positions. In addition, side chains capable of interacting with polar lipid head groups may also be introduced to ensure its proper positioning at the lipid surface. Amide nitrogen alkylation in addition to its hydrogen bonding disruptive capability, also serves another important role, i. e. , it provides a simple strategy for designing gated membrane channels. In general, a wide variety of bi-or multi-dentate small-molecule receptors may be introduced at the channel entrance through N-alkylation. In addition, as discussed previously, these molecules can be capped with molecules similar to BNPA that provide for photoswitchable gating.

EXAMPLE 9 Peptide Construction The peptide can be synthesized by the solid phase method disclosed by Rovero, P. et al. (1991), Tetrahedran Lett., 32,2639-2642 and characterized by 1H-NMR spectroscopy, elemental analysis, and ion-spray mass spectrometry. Although a variety of conditions may be used in the self-assembly of cyclic peptide tubes, the following procedure has provided the most consistent results. Approximately 25 mg/ml suspension of peptide subunit is clarified by the addition of 2.5 equivalents of NaOH. The resulting peptide solution was centrifuged to remove traces of solid matter and then acidified by the addition of 1/3 volume of 1% trifluoroacetic acid in acetonitrile. Particles of cyclic peptide tubes gradually form as a white suspension over a period of hours. Cyclic peptide tubes may then be collected by centrifugation and washed repeatedly with distilled water to remove excess acids and salts. For electron microscopy and diffraction studies, a suspension of particles of cyclic peptide tubes is sonicated briefly and small is drops applied to glow discharged carbon support films on EM grids. Excess liquid is removed by blotting and the grids frozen <BR> <BR> in liquid ethane slush according to the method disclosed by Adrian, M. et al. , (1984) Nature 308,32-36 and Milligan, R. A. et al., (1984) Ultramicroscqpy 13,1-10. Grids are mounted in a Gatan cold stage and examined in a Philips CM12 electron microscope. The specimen temperature was-175°C during examination and imaging.

Images are recorded at 35000X using strict low dose conditions at various defocus levels. For image analysis, micrographs are converted to optical density arrays using a Perkin-Elmer scanning microdensitometer with spot: and step sizes equal to 2. 86 A at the specimen. Using the SUPRIM program package disclosed by, Schroeter4 J. P. et al. (1992), J. Structural Biology 109,235-247, a number of small areas from a single TEM image are rotationally and translationally aligned and then averaged.

EXAMPLE 10 Synthesis of Linear Peptides: The linear form of the target sequence may be synthesized according to conventional solid-phase methods and then cyclized in solution to furnish the desired cyclic peptide subunit. A method for synthesizing linear peptides is provided as follows: Step A: The C-terminal amino acid residue of the target linear peptide is attached to a PAM resin (pheyyl-acetamido-methyl). Hydroxymethyl PAM resin is a preferred PAM resin.

Prior to use, it is washed 4 times in DMF. An N-Boc-aa. l (N-tert-butoxycarbonyl amino acid) is then linked to the washed PAM resin to form Boc-aa. l-PAM resin.

Linkage is achieved by combining the PAM resin with 4 equivalents of N-Boc-amino acid (D or L), 3. 8 equivalents of HBTU (2- (1H- benzotriazol-1-yl)-1, 1,3, 3- tetramethyluronium tetrafluoroborate, and 6 equivalents of DIEA (N, N- diisopropylethylamine) in DMF. The resultant mixture is then shaken for 1 hour. If the C-terminal amino acid residue (aa. l) of the target linear peptide includes a potentially reactive side group, the side group is first blocked by conventional blocking agent prior to its; attachment to the PAM resin. After reaction is complete, the product PAM resin is washed 3 times in DMF for 1 minute.

EXAMPLE 11 Peptide Capping Because the product mixture will include a component of unreacted PAM resin, the PAM resin then is capped by mixing it with 20 equivalents of trimethylacetic anhydride and 10 equivalents of DIEA in DMF and shaking the resultant mixture overnight. The capped PAM resin bearing an N-Boc-amino acid residue is then washed 21 times in DMF and 3 further times in CH2C2 Step C: The protected amino group of the Boc-aa. l- PAM-resin is then deprotected by treatment with neat TFA to form aa. l-PAM-resin. Step D: The deprotected Boc-aa. l-PAM-resin is then coupled to the second amino acid residue (aa. 2), i. e. , the amino acid residue once removed from the C-terminus of the target linear peptide, to form Boc-aa. 2-aa. l-PAM-resin.

The second amino acid residue (aa. 2) has a chirality opposite the chirality of the C- terminal amino acid residue (aa. 1), i. e. , if aa. 1 has a D chirality, aa. 2 has an L chirality; if aa. l has an L chirality, aa. 2 has an D chirality. The deprotected Boc- amino acid-PAM-resin of step C is combined with 4 equivalents of N-Boc-aa. 2,3. 8 equivalents ofHBTU (2- (lH-benzotriazol-l-yl)- 1, 1,3, 3-tetramethyluronium

tetrafluoroborate), and 6 equivalents of DIEA (N, N-diisopropylethylamine) in DMF.

The reaction mixture is then shaken for one hour.

EXAMPLE 12 Peptide Deprotection Step E: The protected amino group of the Boc-aa. 2- aa. l-PAM-resin is then deprotected by treatment with neat TFA to produce aa. 2-aa. l-PAM-resin. Step F: Steps D and E are then repeated as required to couple the third and subsequent amino acid residues in succession to the nascent peptide chain to form a reaction product having the structure aa. n-aa (n-1)-.... aa. l-PAM-resin. The chirality of the even amino acids is opposite the charity of the odd amino acids.

Step G: After the synthesis of the target linear peptide is complete, it is cleaved from the PAM resin. Cleavage is achieved by treatment of the PAM-resin with a 10: 1: 0.5 mixture of HF, anisole, and dimethylsulfide at 0° C for 1 hour. The cleavage product may then be extracted from the reaction mixture with aqueous acetic acid (50% v/v) <BR> <BR> and lyophilized. If the target linear peptide includes protected side! groups, 'these side groups may be deprotected at this time. The product may then be verified by mass spectrometry.

EXAMPLE 13 Synthesis of Selectively N-Alkylated Linear Peptides: The linear form of selectively N-alkylated target peptides may be synthesized according to a modification of conventional solid-phase methods of peptide synthesis.

The linear form of selectively N-alkylated target peptides are then cyclized in solution to furnish the desired selectively N-alkylated cyclic peptide. The method for synthesizing linear form of selectively N-alkylated target peptides employs selectively N-alkylated N-Boc-amino acids. Preferred methods for synthesizing these N-alkylated amino acids and selectively N-alkylated linear peptides are provided as follows: N- Alkylated amino acids may be synthesized according to the method of S. T. Cheung et al. (Canadian Journal of Chemistry (1977), vol. 55, p 906; Canadian Journal of Chemistry, (1977), vol. 55, p 911; and Canadian Journal of Chemistry (1977), vol. 55, <BR> <BR> p 916. ) Briefly, 8 equivalents of methyl iodide were combined with tetrahydrofuran (THF) at 0° C under nitrogen and stirred to form a suspension. Other alkyl iodides and

alkyl bromides may be substituted for the methyl iodide. To this suspension was added 1 equivalent of N-Boc-aa (N-tert-butoxycarbonyl amino acid) as a solid and 3 equivalents of sodium hydride. The resulting mixture was then stirred at room temperature under nitrogen for 24 hours. After 24 hours, excess NaH was quenched by the careful addition of an H20 to the reaction mixture. The mixture was then evaporated and the oily residue partitioned between Et2O and water. The Et2O layer was then washed with aqueous NaHC03. The combined aqueous extracts were then acidified to pH 3 with aqueous citric acid (5%). The acidified product was then extracted into EtOAc. The combined EtOAc layer was then serially washed with H2O, aqueous sodium thiosulfate H20, and brine. The product was then dried over MgS04 and subsequently recrystallized. A typical yield is 86%. Selectively N-alkylated linear peptides may be synthesized by a modification of the method provided above if or the synthesis of non-N-methylated linear peptides. N-Alkylated N-Boc-amino acids are less reactive with respect to coupling reactions as compared to non-N-alkylated N- Boc amino acids. As a consequence, coupling reactions involving N-alkylated N-Boc- amino acids may be less efficient. Accordingly, in order to achieve a high over all yield, it is often useful to follow up each coupling reaction with one or more recoupling reactions.

EXAMPLE 14 A selectively N-alkylated target peptide may be synthesized as follows: Step A: The C-tenninal amino acid residue (aa. l) of the target linear peptide is attached to a PAM resin (phenyl-acetamido-methyl). Hydroxymethyl PAM resin is a preferred PAM resin. Prior to use, it is washed 4 times in DMF. A N-Boc-aa. 1 (N- tert-butoxycarbonyl amino acid) or a N-alkylated N-Boc-aa. l is then linked to the washed PAM resin to form Boc-aa. 1-PAM resin. Linkage is achieved by combining the PAM resin with 4 equivalents of N-Boc-aa. 1 (D or L) or N-alkylated N-Boc-aa. 1 (D or L), 3. 8 equivalents of HBTU (2- (1H- benzotriazol-i-yl)-1, 1,3, 3- tetramethyluronium tetrafluoroborate), and 6 equivalents of DIEA (N, N- diisopropylethylamine) in DMF. The resultant mixture is then shaken for 1 hour. If the C-terminal amino acid residue (aa. 1) of the target linear peptide includes a potentially reactive side group, the side group is first blocked by conventional blocking agent prior to its attachment to the PAM resin. After reaction is complete, the product PAM resin is washed 3 times in DMF for 1 minute.

Step B: Because the product mixture will include a component of unreacted PAM resin, the PAM resin then is capped by mixing it with 20 equivalents of trimethylacetic anhydride and 10 equivalents of DIEA in DMF and shaking the resultant mixture overnight. The capped PAM resin bearing an N-Boc-amino acid residue or N-alkylated N-Boc amino acid is then washed 3 times in DMF and 3 further times in CH2C12.

Step C: The protected amino group of the Boc-aa. 1- PAM-resin or N-alkylated N- Boc-aa. l-Pam resin is then deprotected by treatment with neat TFA to form aa. l- PAM-resin or N-alkylated aa. l-PAM resin, respectively.

Step D: The deprotected Boc-aa. l-PAM-resin or N-alkylated aa. l-PAM resin is then coupled to the second amino acid residue (aa. 2 or N-alkyl aa. 2), i. e, the amino acid residue once removed from the C-terminus of the target linear peptide, to form Boc- aa. 2-aa. l-PAM- resin, Boc-aa. 2-N-alkyl aa. l-PAM-resin, N-alkyl Boc-is aa. 2-aa. l- PAM-resin, or N-alkyl Boc-aa. 2-N-alkyl aa. l- PAM-resin. The second amino acid residue (aa. 2) has a chirality opposite the chirality of the C-terminal amino acid residue (aa. l), i. e. , if aa. l has a D chirality, aa. 2 has an L chirality; if aa. 1 has an L chirality, aa. 2 has an D chirality. The deprotected Boc-aa. l-PAM-resin or N-alkyl Boc-aa. l-PAM-resin of step C is combined with 4 equivalents of N-Boc-aa. 2 or N- alkyl N-Boc-aa. 2,3. 8 equivalents of HBTU (2- (IH- benzotriazol-i-yl)-1, 1,3, 3- tetramethyluronium tetrafluoroborate), and 6 equivalents of DIEA (N, N- diisopropylethylamine) in DMF. The reaction mixture is then shaken for one hour. If coupling is occurring with N-alkyl Boc-aa. l-PAM-resin, the efficiency of the initial coupling reaction may be relatively low. In this event, an aliquot of the reaction mixture may then be assayed by the chloranil test. If the test is positive, the reaction product is treated a second time with the above reactants to achieve an essentially quantitative yield of Boc-aa. 2-N-alkyl aa. l-PAM-resin or N-alkyl Boc-aa. 2-N-alkyl aa. l-PAM-resin.

Step E: The protected amino group of the product of Step D, i. e. , Boc-aa. 2-aa. l-PAM- resin, Boc-aa. 2-N- alkyl aa. l-PAM-resin, N-alkyl Boc-aa. 2-aa. l-PAM-resin, or N- alkyl Boc-aa. 2-N-alkyl aa. l-PAM-resin, is then deprotected by treatment with neat

TFA to produce aa. 2-aa. l-PAM-resin, aa. 2-N-alkyl aa. l-PAM-resin, N-alkyl-aa. 2- aa. 1-PAM-resin, or N-alkyl-aa. 2-N-alkyl aa. 1-PAM-resin.

Step F: Steps D and E are then repeated as required to couple the third and subsequent amino acid residues in succession to the nascent peptide chain to form a target selectively N-alkylated linear peptide linked to resin.

Step G: After the synthesis of the target selectively N-alkylated linear peptide is complete, it is is cleaved from the PAM resin. Cleavage is achieved by treatment of the PAM-resin with a 10: 1: 0.5 mixture of HF, anisole, and dimethylsulfide at 0° C for 1 hour.

The cleavage product may then be extracted from the reaction mixture with aqueous acetic acid (50% v/v) and lyophilized. If the target selectively N-alkylated linear peptide includes protected side groups, these side groups may be deprotected at this time. The product may then be verified by mass spectrometry.

EXAMPLE 15 Synthesis of Linear Peptide Precursors of Gated Cyclic Peptides: A variety of methods may be used to gate the nanotubes of the present invention. The linear form of selectively N-substituted target: peptides may be synthesized according to a modification of conventional solid-phase methods of peptide synthesis. Gated cyclic peptides can be formed by cyclization of linear peptides selectively N- substituted with respect to their peptide backbone amino groups. Each of the preferred substitutions includes a heterocyclic structure linked via an alkyl chain, viz. N- (CHY - heterocycle, where N is a peptide amino nitrogen and"n"lies between 1 and 5. The distal end of the alkyl chain is bonded to a selected peptide amino nitrogen on the peptide backbone. hi an embodiment, all N-substitutions are on the same face of the cyclic peptide. Heterocyclic structures include imidazole, pyridinq, 2, 2 : 6,2" terpyridine, and 2, 2-bipyridine. N-substituted N-Boc-amino acids are employed for synthesizing the linear form of selectively N-substituted target peptides. The method of S. T. Cheung et al. (Canadian Journal of Chemistry (1977), vol. 55, p 906; Canadian Journal of Chemistry (1977), vol. 55, p 911; and Canadian Journal of Chemistry (1977), vol. 55, p 916.) may be employed for synthesizing the N-substituted amino

acids. The synthetic method employs a haloalkyl-heterocycle as a substrate, i. e. , X- (CH2) n-, heterocycle, where X is a halogen and"n"lies between 1 and 5. Preferred halogens include bromine and iodine. Preferred alkyl groups include (CH2) n, where n lies between 1 and 5. The halogen is positioned at one end of the alkyl chain distal with respect to the attachment of the alkyl chain to the heterocycle. Haloalkyl- heterocyclic substrates may be obtained as follows: 4- (Bromomethyl)-l-H imidazole may be synthesized according to the method of D. E. Ryono et al. in German Patent DE 3309014 (09/29/83), claiming priority from US Patent Application Serial No.

356941 (03/15/82) or according to the method of W. Schunack in Arch. Pharm.

(1974), vol. 307 (1), pages 46-51. 4- (2-Bromoethyl)-l-H imidazole may be synthesized according to the method of E. T Chen in Anal. Chem. (1993), vol. 65 (19), pages 2563- 2567. 4- (3-Bromopropyl)-l-H imidazole may be synthesized according to the method of P. Franchetti et al. in Farmaco, Ed. Sci. , vol. 29 (4), pages 309-316 and according to the method of W. M. P. B. Menge et al. in J. Labelled Compd. Radiopharm. (1992), vol.

31 (10), pages 781-786. 3- (Bromomethyl)-pyridine may be synthesized according to the method of R. Jokela et al. in Heterocycles 1985, vol. 23 (7), pages 1707-22. 3- (2- Bromoethyl) -pyridine may be synthesized according to the method of A. Lochead et al. in European Patent Application No. EP 320362 (06/14/89) and EP 88-403079 (12/06/88), claiming priority from French patent application FR 87-17044 or according to the method of R. A. R. Pruneau et al in European Patent application EP 284174 (09/28/88) andEP88-300281 (01/14/88), claiming priority from EP 67-400122 (01/19/87) and EP 87-401798 (07/31/87). 3- (3-Bromopropyl)-pyridinemaybe synthesized according to the method of A. W. Van der Made et al. in Recl. Trav.

Chim. Pays-Bas (1990), vol. 109 (11), pages 537-551. 3- (4-Bromobutyl)-pyridine may be synthesized according to the method of J. W. Tilley et al. in the Journal of Coganic Chemistry (1987), vol. 52 (12), pages 2469-2474 or according to the method of U. R. Patel in U. S. Patent No. 4, 855, 430 (08/08/89) or according to the method of M. Carson et al. in U. S. Patent No. 4,663, 332 (05/05/87). 3- (Iodomethyl)-pyridine may be synthesized according to the method of G. G. Abashev in USSR Patent No.

SU 1692985 Al (11/23/91). 41- (4-Bromobutyl)-2, 2' : 6, 2"-terpyridine may be synthesized according to the method of J. K. Bashkin in PCT International Patent Application No. WO 9119730 Al (12/26/91) or WO 91-US3880 (06/03/91). 5- (Bromomethyl)-2, 2-bipyridine may be synthesized according to the method of J.

Uenishi et al. in the Journal of Organic Chemistry (1993), vol. 58 (16), pages 4382-

4388 or according to the method of B. Imperiali et al. in the Journal of Organic Chemistry (1993), vol. 56 (6), pages 1613-1616. Briefly, 8 equivalents of a haloalkyl- heterocyclic substrate, as indicated above, is combined with tetrahydrofuran (THF) at 0° C under nitrogen and stirred to form a suspension. To this suspension is added 1 equivalent of N-Boc-aa (N-tert-butoxycarbonyq amino acid) as a solid and 3 equivalents of sodium hydride. The resulting mixture is then stirred at room temperature under nitrogen for 24 hours. After 24 hours, excess NaH is quenched by the careful addition of an H20 to the reaction mixture. The mixture is then evaporated and the oily residue partitioned between Et2O and water. The Et2O layer is then washed with aqueous NaHC03-The combined aqueous extracts are then acidified to pH 3 with aqueous citric acid (5%). The acidified product is then extracted into EtOAc. The combined EtOAc layer is then serially washed with H20 aqueous sodium thiosulfate, H20, and brine. The product is then dried over MgS04 and subsequently recrystallized. Selectively N-substituted linear peptides may be synthesized according to the method provided above for the synthesis of N-alkylated or N-methylated linear peptides. If an N-substituent is bulky, the N-substituted N-Boc-amino acids can be even less reactive with respect to coupling reactions than N-methyl N-Boc amino acids due to steric hinderance. As a consequence, coupling reactions involving N- substituted N-Boc-amino acids may be slow and relatively inefficient. Accordingly, in order to achieve a high over all yield, it is often useful to follow up each coupling reaction with repeated recoupling reactions.

EXAMPLE 16 A selectively N-substituted target peptide may be synthesized as follows: Step A: The C-terminal amino acid residue (aa. l) of the target linear peptide is attached to a PAM resin V (phenyl-acetamido-methyl). Hydroxymethyl PAM resin is a preferred PAM resin. Prior to use, it is washed 4 times in DMF. A N-Boc-aa. 1 (N- tert-butoxycarbonyl amino acid) or a N-substituted N-Boc-aa. 1 is then linked to the washed PAM resin to form Boc-aa. l-PAM resin. Linkage is achieved by combining the PAM resin with 4 equivalents of N-Boc-aa. 1 (D or L) or N-substituted N-Boc-aa.

1 (D or L), 3.8 equivalents of HBTh (2-(lH-benzotriazol-l-yl)-1, 1,3, 3- tetramethyluronium tetrafluoroborate), and 6 equivalents of DIEA (N, N- diisopropylethylamine) in DMF. The resultant mixture is then shaken for 1 hour. if the C-terminal amino acid residue (aa. l) of the target linear peptide includes a

potentially reactive side group, the side group is first blocked by conventional blocking agent prior to its attachment to the PAIM resin. After reaction is complete, the product PAM resin is washed 3 times in DMF for I minute.

Step B: Because the product mixture will include a component of unreacted PAM resin, the PAM resin then is capped by mixing it with 20 equivalents of trimethylacetic anhydride and 10 equivalents of DIEA in DMF and shaking the resultant mixture overnight. The capped PAM resin bearing an N-Boc-amino acid residue or'N-substituted N-Boc amino acid is then washed 3 times in DMF and 3 further times in CH2C12 Step C: The protected amino group of the Boc-aa. 1-PAM-resin or N-substituted N- Boc-aa. l-Pam resin is then deprotected by treatment with neat TFA to form aa. l- PAM-resin or N-substituted aa. 1-PAM resin, respectively.

Step D: The deprotected Boc-aa. l-PAM-resin or N-substituted aa. 1-PAM resin is then coupled to the second amino acid residue (aa. 2 or N-alkyl aa. 2), i. e. , the amino acid residue once removed from the C-terminus of the target linear peptide, to form Boc-aa. 2-aa. l- PAM-resin, Boc-aa. 2-N-alkyl aa. l-PAM-resin, N-alkyl t Boc-aa. 2- aa. l-PAM-resin, or N-alkyl Boc-aa. 2-N-alkyl aa. l-PAM-resin. The second amino acid residue (aa. 2) has a chirality opposite the chirality of the C-terminal amino acid residue (aa. l), i. e. , if aa. l has a D chirality, aa. 2 has an L chirality ; if aa. l has an L chirality, aa. 2 has an D chirality. The deprotected Boc-aa. l-PAM-resin or N-alkyl Boc-aa. l-PAM-resin of step C is combined with 4 equivalents of N-Boc-aa. 2 or N- alkyol N-Boc-aa. 2,3. 8 equivalents of HBTU (2- (1H- is benzotriazol-i-yl)-1, 1,3, 3- tetramethyluronium tetrasfluoroborate), and 6 equivalents of DIEA (N, N- diisopropylethylamine) in DMF. The reaction mixture is then shaken for one hour. If coupling is occurring with N-alkyl Boc-aa. l-PAM-resin, the efficiency of the initial coupling reaction may be relatively low. In this event, an aliquot of the reaction mixture may then be assayed by the chloranil test. If the test is positive, the reaction product is treated second time with the above reactants to achieve an essentially quantitative yield of Boc-aa. 2-N-alkyl aa. l-PAM-resin or N-alkyl Boc-aa. 2-N-alkyl aa. l-PAM-resin.

Step E: The protected amino group of the product of Step D, i. e. , Boc-aa. 2-aa. l-PAM- resin, Boc-aa. 2-N- alkyl aa. l-PAM-resin, N-alkyl Boc-aa. 2-aa. l-PAM-resin, or N- alkyl Boc-aa. 2-N-alkyl aa. l-PAM-resin, is then deprotected by treatment with neat TFA to produce aa. 2- aa. l-PAM-resin, aa. 2-N-alkyl aa. l-PAM-resin, N-alkyl-aa. 2- aa. l-PAM-resin, or N-alkyl-aa. 2-N-alkyl aa. l-PAM- resin.

Step F: Steps D and E are then repeated as required to couple the third and subsequent amino acid residues in succession to the nascent peptide chain to form a target selectively N-substituted linear peptide linked to resin.

Step G: After the synthesis of the target selectively N-substituted linear peptide is complete, it is cleaved from the PAM resin. Cleavage is achieved by treatment of the PAM-resin with a 10: 1: 0.5 mixture of HF, anisole, and dimethylsulfide at 0° C for 1 hour.

The cleavage product may then be extracted from the reaction mixture with aqueous acetic acid (50% v/v) and lyophilized. If the target selectively N-substituted linear peptides includes protected side groups, these side groups may be deprotected at this time. The product may then be verified by mass spectrometry.

EXAMPLE 17 Cyclization of Linear Peptides: The target linear peptides, selectively N-alkylated target linear peptides, and selectively N-substituted target linear peptides, whose syntheses are described above, may each be cyclized according to the following protocol: A solution of the linear peptide in DMF (1 mM) is treated with TBTU (2-(lH-Benzotriazol-l-yl)-1, 1,3, 3- tetramethyluroniuam tetrafluoroborate, 3 mM, HOBt (1-Hydroxybenzotriazole, 3 mM) and DIEA (diisopropylethylamine, 1% v/v) at 5° C for 12 hours to give the desired cyclic peptide monomer. The product may be purified by reverse-phase HPLC purification. A typical yield for the cyclization of N-methylated linear peptides octomer is 70% yield.

EXAMPLE 18 Alternative Method for Peptide Synthesis and Cyclization: Alternatively, peptides containing an Asp residue can be synthesized and cyclized by the solid phase method disclosed by Rovero, P. et al. (1991), Tetrahedron Lett. , 32, 2639-2642. Briefly, Boc-Asp (N-tert-butoxycarbonyl aspartic acid) is linked to PAM resin (phenyl-acetamido-methyl) through the B carboxylic function while the a- carboxylic groups is protected as as fluorenylmethyl ester (OFm). Boc-Asp (13- PAM-resin) OFm may be purchased from Bachem AG, Switzerland. A B linear peptide having the D-L chirality motif may then be built upon the Boc-Asp (B-PAM- resin) OFm according to the classical Boc/Benzyl strategy using an automatic or semi- automatic peptide synthesizer--e. g. Labortec SP 640. Synthesis is achieved by consecutively adding Boc-protected amino acids according to the BOP coupling <BR> <BR> procedure, i. e. , 3 equivalents Boc-amino acid, 3 equivalent BOP and 6 is equivalent DIEA, in DMF for 1 hour. Completeness may be achieved by repeating each coupling twice. Once the synthesis of the linear peptide is complete, it is ready for cyclization.

Prior to cyclization, the N-terminal amino group was deprotected with TFA and the C-terminal fluorenylmethyl ester was deblocked with piperidine (20% v/v piperidine in DMF) for 3 + 7 minutes. Cyclization was then achieved by treatment with 3 equivalents of BOP (benzotriazolyl-N-oxy-tris (dimethylamino)-phosphonium hexafluorophosphate) and 6 equivalents of DIEA (N, N-diisopropylethylamine) in DMF for 3 hours. If the cyclization reaction is incomplete, the BOP treatment may be repeated. Deprotection of the side chains and cleavage of the cyclic peptides from the resin may be achieved by treatment with a 10: 1: 0.5 mixture of HF, anisole, and dimethylsulfide at 0° C for 1 hour. The product may then be extracted from the reaction mixture with aqueous acetic acid (50% v/v) and lyophilized. Capped and gated cyclic peptides may also be synthesized according to the above method by cyclizing the corresponding N-substituted linear peptides.

EXAMPLE 19 BNPA: a water soluble reagent for"caging"sulfhydryls.

The photoremovable 2-nitrobenzyl group has been used for the protection of many functionalities in synthetic organic chemistry and for the production of cage reagents in biology. Relevant to the present nanotubes, the sulfhydryl group in the cysteine has been protected with 2-nitrobenzylchloride as S-2-nitrobenzylcysteine for the

applications in peptide synthesis. Cysteine containing peptides were generated from protected derivatives in good yields by irradiation at &gt; 350 nm, provided that reagents were present to trap the photoproduct 2-nitrosobenzaldehyde and to prevent sulfhydryl oxidation. In attempt to protect preformed cysteine containing peptides and proteins, we found that nitrobenzyl chloride and related commercial reagents were too insoluble for aqueous buffers to permit efficient derivatization under most circumstances. Therefore, a different molecule 2-bromo-2- (2-nitrophenyl) acetic acid (BNPA) was created that introduced the a-carboxy-2-nitrobenzyl (CNB) protecting group. The molecule can be produced in high yield by the bromination of 2- nitrophenylacetyl chloride, followed by hydrolysis of the acyl chloride group (See FIG. 2 of Chang et al., Chemistry & Biology, 1995, Vol. 2 No. 6, herein incorporated by reference in its entirety). BNPA is a highly water-soluble molecule at pH values around neutral, and the a-carboxyl group increases the reactivity of the electrophilic center towards the cysteine anion. Further, the presumed photoproduct, 2- nitrosoglyoxylic acid is less reactive than the 2-nitrosobenzaldehyde photoproduct. In addition, small peptides or nanotubes containing the CNB group should be more soluble in water than those protected with the simple 2-nitrobenzyl group. FIG. 6 and FIG. 7 show how the nanotubes may be gated to switch"open"when the photreactive group is removed by the addition of light to the composition. The steric hindrance provided by the large BNPA molecules block the end of the nanotube. Upon irradiation at > 300 nm the RS-CNB is converted to RSH and a degradable product.

Synthesis of 2-bromo-2- (2-nitrophenyl) acetic acid (BNPA) The general procedure for a-halogenation of acyl chlorides described by Harp et al <BR> <BR> was used (Harp et al. , An efficient halogenation of acyl chlorides by N-bromo- succinimide, N-chlorosuccinimide, and molecular iodine. J. Org. Chem. 40,3420- 3427. To 2- (2-nitrophenyl) acid acid (Aldrich, 5.0 g, 27.6 mmol) was added carbon tetrachloride (5 ml) and thionyl chloride (7.95 mo, 109 mmol). The mixture was stirred at 65 °C for 1.5 hours to form the acyl chloride, after which N- bromosuccinimide (5.90 g, 33.1 mmol), CC14 (25 ml) and a catalytic amount (11 drops) of HBr in acetic acid were added to the flask. The mixture was heated at 70 °C.

After 4.5 hours, ice (25 g) was added to the cooled mixture, which was stirred vigorously for 1 hour to hydrolyze the acyl chloride. The CCl4 layer was retained and

aqueous phase was extracted with 3 x 25 ml CH2C12. The combined organic fractions where dried with Na2S04 and the solvent was removed by evaporation furnishing crude 2-bromo-2- (2-nitrophenyl) acetic acid in near quantitative yield as a brown oil.

For techniques on further characterization and recrystallization, see Chang et al., Chemistry and Biology, June 1995,2 : 391-400.

BNPA modification of nanotube capped peptides In order for the BNPA to react with the nanotube peptides, the cyclic peptide needs to be n-alkylated or thioalkylated. This can be accomplished in a variety of methods known in the art and described above. The function is to prepare the peptide with appropriate RSH functionalities that may then reacted with the BNPA molecules to form the RS-CNB compositions (See FIG. 6 and FIG. 7). The nanotubes were then reduced for 5 minutes using 10 mM DTT, approximately 1.0 M Tris HC1, pH 8.5 and water. 100 mM BNPA in 100 mM NaPi, pH 8.5 was then added and the modification reactions wee incubated at room temperature in the dark for approximately 1-9 hours.

At the end of the reaction excess BNPA and its low mass sulfhydryl adducts (e. g. with DTT) were removed by repeated cycles of dilution with 100 mM Tris HC1 (pH 6.0 or 8.5) and concentration by ultrafiltration (Amicon, Microcon-3). The final volume was readjusted with 100 mM Tris HC1 containing 10 mM DTT so that the concentration of DTT was 1 mM. The final concentration of BNPA and its low mass sulfhydryl adducts was < 20 p1M. The modified peptides were stored at-20 °C before use.

Photolysis of nanotube (caged peptides) CNB-RS in 100 mM Tris HC1 (pH 6.0 or 8.5) containing 1 mM DTT was placed in a well of 96 microtiter plate and irradiated for 30 minutes on ice through a 285 nm cut off filter (Oriel #51220) at 3.5 cm from a Foto UV 300 illuminator. To assay for the unmasking of the protected cysteine residue, samples of the irradiated proteins were treated with IASD and analyze using SDS-PAGE. For further details see Chang et al., Chemistry & Biology, 1995, Vol. 2 No. 6.

EXAMPLE 20 Manufacturing of photactivatable PEG surface graft coated liposomes The manufacturing and assembly of the therapeutic composition may be accomplished using a variety of methods. The manufacture of the lipids begins when the phospholipids, including the PEG or MPEG based liposomes are first dissolved in a water-miscible organic solvent such as ethanol. This solution is then injected into an aqueous solution, which dilutes the organic solvent and the lipid molecules spontaneously arrange themselves to form liposomal structures, capturing the surrounding aqueous medium within the internal aqueous compartment of the liposomes. At this step (hydration), the liposomes are very large and heterogeneous.

The liposome suspension is then put through a size reduction step to obtain homogenous, small-sized liposome preparation. Size reduction can be achieved with high-pressure homogenization techniques or extrusion through track-etched membranes of defined pore size.

Therapeutic agents such as drugs and nanotubes can be added to the liposomes at any point. However, it may be advantageous to first drug load the nanotubes and then assemble them in the membranes. Alternatively, the nanotubes can be assembled in the membranes and then drug loaded. The drug loading lowers overall leakage of drugs added during active loading. After the photactivatable nanotubes have been positioned in the liposomes, they may be tested for leakage in a variety of ways know in the art. Potential leakage problems and issues may be addressed by adding one or more subsequent lipid layers as shown in FIG. 2 and FIG. 2A. Next, active loading may be employed to actively load drugs such as daunorubicin or doxorubicin to the liposomes. In this case, the hydration step is performed to encapsulate an ammonium sulfate solution. After size reduction, the extraliposomal ammonium sulfate is removed by diafiltration. Doxorubicin or other drugs may then be added to the liposome preparation. The absence of ammonium sulfate in the extraliposomal phase establishes a chemical gradient that induced the drug to diffuse into the liposomes and become entrapped inside.

Active loading is usually more efficient than passive loading. More than 90% of the added drug becomes encapsulated during the loading of the drug, while typical efficiency of passive loading ranges from 20% to 40%. After loading, the

unencapsulated drug can be removed by diafiltration or ion exchange methods of needed. The preparation may then be sterilized by passage through a 0.2 llm sterilization membrane and filled into final product vials. When needed the product can by lyophilized for added stability. The complex manufacturing process usually takes several days to complete. Depending on drug potency, production scale at early clinical development stages varies from a few liters to tens of liters, while commercial scales may range from fifty to several hundred liters. Product characteristics (e. g. drug potency, nanotube design, lipid composition, liposome size, capping of nanotubes, drug loading of nanotubes, percent drug encapsulation, drug to lipid ratio, nanotube to lipid ratio, and drug leakage) need to be determined early on in the process.